Strand displacement stop (sds) ligation

ABSTRACT

A method of ligating DNA molecules, wherein the DNA molecules are in a hybrid with an RNA molecule, including the steps of providing DNA molecules that are in a RNA:DNA hybrid with an RNA molecule, and ligating the DNA molecules to each other with a double strand specific ligase

CROSS REFERENCE TO RELATED APPLICATIONS

This is continuation of U.S. patent application Ser. No. 14/241,311,which is a national phase patent application under 35 U.S.C. §371 ofinternational patent application serial no. PCT/EP2012/068250, filedSep. 17, 2012, which claims priority to European patent applicationserial no. 11181546.0, filed Sep. 16, 2011 and European patentapplication serial no. 12177647.0 filed Jul. 24, 2012; the contents ofeach are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of amplifying or analyzingsamples of nucleic acids by amplification of defined sequence portions.

BACKGROUND OF THE INVENTION

Numerous amplification-based methods for the amplification and detectionof target nucleic acids are well known and established in the art. Thepolymerase chain reaction, commonly referred to as PCR, uses multiplecycles of denaturation, annealing of primer pairs to opposite strands,and primer extension to exponentially increase copy numbers of thetarget sequence (U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; U.S.Pat. No. 4,800,159; U.S. Pat. No. 5,804,375). In a variation calledRT-PCR, reverse transcriptase (RT) is used to make a complementary DNA(cDNA) from RNA, and the cDNA is then amplified by PCR to producemultiple copies of DNA (U.S. Pat. No. 5,322,770; U.S. Pat. No.5,310,652).

PCR reactions generally comprise carrying out multiple cycles of:

(A) hybridizing (annealing) a first primer to a site in a nucleic acidstrand at one end of the target nucleic acid sequence, and hybridizing asecond primer to a site corresponding to the opposite end of the targetsequence in the complementary nucleic acid strand;(B) synthesizing (extending) a nucleic acid sequence from eachrespective primer; and(C) denaturing the double stranded nucleic acid produced in step (B) soas to form single stranded nucleic acid.Denaturation is generally carried out at from 80 to 100° C.,hybridization (annealing) is generally carried out at from 40 to 80° C.,and extension is generally carried out at from 50 to 80° C. A typicalcycle is denaturation: about 94° C. for about 1 min, hybridization:about 58° C. for about 2 min, and extension: about 72° C. for about 1min. The exact protocol depends on factors such as the length andsequence of the primers and target sequence, and the enzyme used.

PCR has been adopted for various applications. E.g., GB 2293238describes methods to reduce non-specific priming and amplifying nucleicacid sequences. Blocking “primers” (or oligonucleotides) are disclosedthat produce misalignment and reduce non-specific priming by creatingcompetitive primer annealing reactions to the amplification primers. Forexample, mixtures of random blocking primers are used that comprise addNTP at the 3′ position to prevent initiation of extension reactions.Only correct amplification primers displace their blocking primers andcan initiate the amplification reaction.

Methods have been established for using blocking primers thatspecifically bind to unwanted target oligonucleotide molecules in asample to prevent amplification thereof in a PCR reaction of unblockedoligonucleotide molecules. Unblocked oligonucleotides can be amplifiedwithout further measures to ensure target specificity—in the absence ofamplifiable competitive oligonucleotide molecules that are not intendedfor amplification (US 2002/0076767 A1 and U.S. Pat. No. 6,391,592 B1; WO99/61661).

A similar method is disclosed in the WO 02/086155, wherein blockingoligonucleotides bound to an undesired template result in prematuretermination of an elongation reaction. The blocking oligonucleotidesbind specifically to one template in a mixture while leaving othertemplates free for amplification.

U.S. Pat. No. 5,849,497 describes the use of blocking oligonucleotidesduring a PCR method with a DNA polymerase lacking 5′ exonucleaseactivity. This DNA polymerase cannot digest the blockingoligonucleotides that prevent amplification. Such a system has beenselected to avoid using PNA (peptide nucleic acids) as blockingoligonucleotides. A similar system is described in WO 2009/019008 thathowever contemplates the use of PNA and LNA, among others, as blockingoligonucleotides.

All these methods have in common that amplification of unwantedtemplates is specifically suppressed by hybridization of a specificblocking oligonucleotide.

In patent application WO 98/02449 A1 (U.S. Pat. No. 6,090,552) a“triamplification” DNA amplification method is described. It is based onthe use of a hairpin primer that is extended and ligated to a blocker.Both primer and blocker bind to one template DNA strand. The secondprimer binds to the complementary DNA strand. The blocker and one primerare partially complementary with the primer containing a donor and theblocker containing an acceptor moiety for FRET (Fluorescence ResonanceEnergy Transfer). An extension of the primer and a ligation of anextension product to the blocker leads to a decrease in fluorescence,because they are no longer in close proximity in a blocker primerhybrid. This triamplification method is limited to the use of templateDNA and does not relate to RNA methods.

WO 94/17210 A1 relates to a PCT method using multiple primers for bothanti-sense and sense strand of a target DNA.

Seyfang et al. [1] describe the use of multiple phosphorylatedoligonucleotides in order to introduce mutations into a DNA strand. T4DNA polymerase, which lacks any detectable strand displacement activityor 5′-3′ exonuclease activity, is used, which is unsuitable for RNAtemplates.

Hogrefe et al. [2] describe the generation of randomized amino acidlibraries with the QuikChange Multi Site-Deirected Mutagenisis Kit.Specific primers containing 3 degenerate nucleotides in the centercomplementary to a known single stranded target DNA are used. Thedescribed kit uses PfuTurbo DNA polymerase which is usuitable for RNAtemplates.

The analysis of RNA regularly starts with reverse transcribing RNA intocDNA as DNA is more stable than RNA and many methods exist for analyzingDNA. Whatever protocol is used to analyze the cDNA, it is important thatthe cDNA generated during reverse transcription (RT) represents the RNAthat needs to be analyzed in sequence and concentration as closely aspossible.

Reverse transcription is generally carried out using reversetranscriptases. These enzymes require an oligonucleotide primer thathybridizes to the RNA to start (prime) the template dependentpolymerization of the cDNA. The two most common priming strategies usedare oligo dT priming and random priming.

Oligo dT priming is used for RT of mRNAs that have a poly A tail ontheir 3′ end. The oligo dT primes the RNA at the 3′ end and the reversetranscriptase copies the mRNA up to its 5′ end. One drawback of thisapproach is that high quality mRNA is needed as any mRNA degradationwill lead to a strong overrepresentation of the 3′ ends of mRNAs.

Even if un-degraded mRNA is used the cDNA molecules may still betruncated due to premature polymerization stop events. A frequent causeis secondary and tertiary structure formation in highly structured RNAregions. Especially when the GC content is high the reversetranscriptase might not read through these regions and thus the cDNAbecomes truncated. The likelihood of such events to occur increases thelonger the mRNA is that needs to be copied. Therefore oligo dT primedcDNA can show a strong bias towards over-representing the 3′ ends ofRNAs. Thus, 3′ end priming suffers from a concentration bias that leadsto an increase of sequences at or near the 3′ end with gradually reducedrepresentation of sequences in the direction of the 5′ end (see FIG. 15,triangles, for qPCR measurement of the bias). This is problematic inquantitative approaches, e.g. in the determination of the degree ofexpression of a particular gene, in difference analysis or in completeexpression profiling of a cell.

Approaches have been developed to overcome RNA secondary structuretermination especially when long mRNAs need to be reverse transcribedinto full length cDNAs. One such method for instance involves a mixtureof 2 reverse transcriptases, one highly processive such as MMLV or AMVand mutants thereof, first incubating the reaction mixture at a normaltemperature range to allow first strand synthesis plus using athermostable enzyme composition having reverse transcriptase activityand then incubating the reaction mixture at a temperature that inhibitsthe presence of secondary mRNA structures to generate a first strand(U.S. Pat. No. 6,406,891). However, buffers for reverse transcriptasesontain high concentrations of MgCl₂ ₂ (3-10 mM) or Mn²⁺ (e.g for Tth DNApolymerase) and RNA is highly unstable and susceptible to breaks and/ordegradation at higher temperatures especially in the presence of thesedivalent cations. The cycling method between two temperatures to bypasssecondary structures might also lead to random priming by short RNAfragments that were generated during high temperatures. Such short RNAfragments will be used by MMLV-H or other viral reverse transcriptasesas a primer [3]. Again this would lead to a bias in the synthesizedcDNA.

Another approach is random priming that has the advantage of hybridizingat multiple locations along the RNA and hence also blocking thosesequences from taking part in secondary structure formation. In randompriming an oligonucleotide population of random sequence, usually arandom hexamer is used to prime the RT anywhere within the templatenucleic acid strand. Random priming is used for both, reversetranscription or regular transcription using DNA as template. Whenproduct DNA was analyzed it was found that random priming does notresult in equal efficiencies of reverse transcription for all targets inthe sample [4, 5]. Furthermore there is no linear correlation betweenthe amount of template nucleic acid input and product DNA output whenspecific targets are measured [4, 5]. Indeed, it has been shown that theuse of random primers can lead to overestimate some template copynumbers by up to 19-fold compared to sequence-specific primed templates[6]. Although a lot was speculated about the underlying causes for thesephenomena, no conclusive rational has been put forward.

Objective

The present inventors have observed that there is a general bias in suchrandomly primed cDNA libraries, in the form that the sequence parts onthe 5′ ends of RNA molecules are overrepresented when compared to theparts on 3′ ends. The reason for this phenomenon is to be found in thecombination of random priming and the strong strand displacementactivity of the reverse transcriptases. As RNA has a high degree ofsecondary structure, reverse transcriptases had to evolve a strongstrand displacement activity to overcome this secondary structure and toeffectively generate cDNA. Given the strong strand displacement activityof reverse transcriptase the 5″ side of any RNA will be representedseveral times in a cDNA library when random oligonucleotides are usedfor priming. A similar effect happens during extension of (e.g. random)primer combinations having primers that anneal to a more 3′ position onthe template RNA (upstream primers in the direction of the extensionproducts), wherein the reverse transcriptase will displace the extensionproducts of all primers that have hybridized to a more 5′ position onthe template RNA (downstream primers in the direction of the extensionproducts). Therefore random priming is a DNA synthesis method with astrong bias to over-represent the 5′ ends of a given template nucleicacid (see also FIG. 1 for a schematic representation of the problem andFIG. 15 for qPCR measurements of the bias). Besides using randomers(e.g.: random hexamers) for cDNA library preparation and in radiolabeling of DNA probes [7, 8], they are also used to detect SingleNucleotide Polymorphisms (SNPs) as well as small scale chromosomeevents, primarily insertions or deletions [5, 6]. Comparative GenomicHybridization (CGH) has been developed to elucidate genome-wide sequencecopy-number variation (CNV) between different genomes, such as thedifferential amplification or deletion of genetic regions between tumorDNA and normal DNA from neighboring unaffected tissue [9, 10].

Currently one of the most complete analysis methods for DNA libraries isNext Generation Sequencing (NGS) [for review see 11]. NGS is a genericterm for parallel sequencing through polymerization in a high throughputmanner. NGS is based on obtaining sequencing reads from small fragments.In the generation of cDNA libraries, either the mRNA is fragmentedbefore cDNA synthesis or single stranded or double stranded cDNA isfragmented. However, any fragmentation of template nucleic acids(chemical or physical) introduces an undefined and not foreseeable biasand will deplete the template. In NGS, the complete sequence is obtainedby alignment of those reads which is a challenging task due to the sheernumber of small reads that have to be assembled to a complete sequence.To date many reads provide just limited information. For instance manyof the reads cannot be assigned uniquely and therefore are discarded.Sequence generation is further hindered by representation bias ofsequence fragments.

Therefore there is the need for improved methods for amplifying templatenucleic acids that yield less bias in the amplified amount, e.g. for NGSor for the generation of DNA libraries to improve representation of thesequence concentration of the original template.

SUMMARY OF THE INVENTION

Therefore the present invention provides a method for generating anamplified nucleic acid portion of a template nucleic acid, comprising

obtaining said template nucleic acid,annealing at least one oligonucleotide primer to said template nucleicacid,annealing at least one oligonucleotide stopper to said template nucleicacid,elongating the at least one oligonucleotide primer in a templatespecific manner until the elongating product nucleic acid reaches theposition of an annealed oligonucleotide stopper, whereby the elongationreaction is stopped, wherein in said elongation reaction saidoligonucleotide stopper is not elongated, andwherein the elongated product nucleic acid is labelled at the 3′ end ata position adjacent to said oligonucleotide stopper and/orwherein the elongated product nucleic acid is ligated to the 5′ end ofsaid oligonucleotide stopper; thus obtaining an amplified nucleic acidportion.

In a further aspect the present invention provides a method ofgenerating an amplified nucleic acid of a template nucleic acid,comprising

obtaining said template nucleic acid,annealing a first oligonucleotide primer to said template nucleic acid,annealing at least one further oligonucleotide primer to said templatenucleic acid,elongating said first oligonucleotide primer in a template specificmanner until the elongating product nucleic acid reaches the position ofone of said further oligonucleotide primers, whereby the elongationreaction is stopped, and at least one further oligonucleotide primer iselongated in a template specific manner,wherein the elongated product nucleic acid is labelled at the 3′ end ata position adjacent to said further oligonucleotide primer and/orwherein the stopped elongated product nucleic acid is ligated to the 5′end of said further oligonucleotide primer; thus obtaining an amplifiednucleic acid portion. In this method said further oligonucleotide primerserves both as a stopper, which prevents further elongation of anamplification reaction that reaches the position of the annealedstopper, and as a primer itself, i.e. as an initiator of elongation.

The template nucleic acid can comprise or substantially consist of RNAor DNA, in preferred embodiments said template is RNA.

In a preferred aspect the present invention provides a method ofgenerating an amplified nucleic acid of a template nucleic acid, whichis RNA, comprising

obtaining said template RNA,annealing a first oligonucleotide primer to said template RNA,annealing at least one further oligonucleotide primer to said templateRNA and/or at least one oligonucleotide stopper,elongating said first oligonucleotide primer in a template specificmanner until the elongating product nucleic acid reaches the position ofone of said further oligonucleotide primers or oligonucleotide stopper,whereby the elongation reaction is stopped,wherein in said elongation reaction said optional oligonucleotidestopper is not elongated and/or at least one further oligonucleotideprimer is elongated in a template specific manner. In preferredembodiments the elongated product nucleic acid is ligated to the 5′ endof said oligonucleotide stopper or further primer.

In a further aspect, the present invention provides the use of theinventive methods to generate a sequence library of one or more templatenucleic acids comprising a mixture of, preferably overlapping, amplifiednucleic acid portions of said template nucleic acids. A sequence libraryis a collection of DNA fragments that can be stored and copied throughany process known in the art. For instance a sequence library can beobtained through the process of molecular cloning. Sequence librariescan also be amplified through e.g. PCR using universal sequences on theends of the DNA fragments.

The invention also relates to a kit for generating amplified nucleicacid portions of a template nucleic acid or for generating a sequencelibrary as mentioned above. An inventive kit comprises a reversetranscriptase, random oligonucleotide primers which comprise amodification that increases the Tm (melting temperature) and randomoligonucleotide stoppers that are unsuitable for nucleotide extensionand comprise a modification that increases the Tm, optionally furtherone or more of reaction buffers comprising Mn²⁺ or Mg²⁺, a ligase,preferably a DNA ligase or RNA ligase with DNA ligating activity, PEG.

The following detailed disclosure reads on all aspects and embodimentsof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the principle problem theinvention seeks to solve.

FIG. 2 is a schematic representation of one embodiment of the inventionto create a 5′-3′ balanced cDNA library and full length cDNA.

FIG. 3 is a schematic representation of creating a linker tagged shortcDNA library.

FIG. 4 is a schematic representation of creating a linker tagged shortcDNA library using an alternative stopper oligo concept.

FIG. 5 is a schematic representation of creating a linker tagged shortcDNA library using an alternative stopper oligo concept.

FIG. 6 is a schematic representation of preferred primer modifications.

FIG. 7 is a schematic representation of preferred oligo stoppermodifications.

FIG. 8 is a schematic representation of the most preferred oligo starterand stopper combinations.

FIG. 9 is a schematic representation of the oligo starter and stopperstructures.

FIG. 10 shows stopping of strand displacement during reversetranscription.

FIG. 11 is an imaging depicting regulation of cDNA fragment size byamount of stop oligos inserted into the RT.

FIG. 12 is an image depicting stopping strand displacement duringreverse transcription plus ligation of the cDNA fragments to a fulllength product.

FIG. 13 is an image depicting validation of SDS/Ligation on mRNA.

FIG. 14 is a table and image depicting strand displacement stop duringreverse transcription plus ligation of the cDNA fragments to a fulllength product results in more product of a selected cDNA.

FIG. 15 is a graph depicting cDNA length comparison of SDS/ligation vsoligo dT priming on a 15 kb cDNA.

FIG. 16 is a table and image depicting generation of a di-tagged DNAlibray from mRNA.

FIG. 17 is a graph depicting a discovery blot comparing a librarypreparation using the new strand displacement stop and ligation protocol(SDS-ligation) with a standard mRNA Seq protocol (TRUSEQ) RNA sampleprep kit, Catalog # RS-930-20 01).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for generating an amplifiednucleic acid or amplifying nucleic acids. This generation can relatealso to a single amplification reaction, e.g. one transcription cycle,or more. It includes the generation of RNA or DNA by RNA or DNAdependent polymerization. Thus, amplifying nucleic acids includedpolymerization of RNA nucleotides based on an RNA or a DNA templatenucleic acid or the polymerization of DNA nucleotides based on an RNA ora DNA template nucleic acid. Preferably the method includes one step orcycle of reverse transcription, RNA dependent DNA polymerization.

The inventive methods include the use of at least two shortoligonucleotides that hybridize with the template nucleic acid. At leastone oligonucleotide has a primer function, i.e. it can act as nucleotidepolymerization initiator for polymerase dependent amplification, i.e.transcription. The extension of primers by the addition of nucleotidesin a template dependent fashion is referred herein as elongation orextension. The products of such reactions are called elongation productsor extension products. RNA or DNA polymerases add nucleotides to givenoligonucleotide strand which base pair to a nucleobase of a templatestrand. Hybridization and annealing is understood as base pairing ofcomplementary nucleotides. Complementary nucleotides or bases are thosecapable of base pairing such as A and T (or U); G and C; G and U.

At least one further oligonucleotide has a stopper function. This meansthat as an elongation (extension) reaction that has been initiated at anupstream primer (in the direction of the extension products) reaches adownstream (in the direction of the extension products) oligonucleotidewith a stopper function, said elongation reaction is prevented fromfurther elongation. Relative to the nucleotide position on the templatenucleic acid this means that once an elongation reaction that has beeninitiated from a primer that has annealed more to the 3′ end of thetemplate nucleic acid relative to the oligonucleotide with the stopperfunction, reaches that oligonucleotide (stopper), said elongationreaction is prevented from further elongation. The elongation reactioncan be stopped by strong hybridization of the oligonucleotide withstopper function to the template nucleic acids so that it is notdisplaced by the polymerase.

The oligonucleotide with stopper function can also be a primer. It canhybridize downstream (in the direction of the elongation reaction,upstream in the direction of the template) to a first primer and stopthe elongation reaction of said first primer. In turn (orsimultaneously) it acts as elongation initiator itself to produce atranscription product—that in turn may also be stopped at the positionof a further downstream (in relation to the direction of the elongationreactions) oligonucleotide with stopper function.

“Upstream” relates to the direction towards the 5′ end (3′-5′) of agiven nucleic acid or oligonucleotide. “Downstream” relates to thedirection towards the 3′ end (5′-3′) of a given nucleic acid oroligonucleotide. Since oligonucleotides hybridize in inverse fashiondownstream for a primer relates to the upstream direction of ahybridized template nucleic acid. This means that a downstreamoligonucleotide (or oligo, or primer or stopper or blocker) is an oligothat hybridized to a more upstream portion of a template nucleic acid inrelation to an upstream oligonucleotide (or oligo, or primer or stopperor blocker) that has hybridized to a more downstream portion of atemplate nucleic acid. Therefore, when using the term “downstreamoligonucleotide”, or “upstream oligonucleotide” the directionalityalways refers to that of the extension product(s), except when statedotherwise. The polymerase dependent elongation reactions of theinvention are in 5′-3′ direction, downstream.

As used herein, “comprising” shall be understood as referring to an opendefinition, allowing further members of similar or other features.“Consisting of” shall be understood as a closed definition relating to alimited range of features.

As used herein “primer” may also refer to an oligonucleotide primer.“Stopper” refers also to oligonucleotide stopper. “Oligo” is used forboth oligonucleotide primers and oligonucleotide stoppers.

An oligonucleotide stopper is an oligonucleotide that can stop anelongation reaction as described above and does not initiate a furtherelongation reaction, e.g. the oligonucleotide is incapable of accepting(covalently binding) a further nucleotide at its 3′ position. Suchnucleotides are known in the art and usually lack a 3′ OH, as e.g. inddNTS (dideoxynucleotides).

Thus, the present invention essentially provides two methods, oneutilizing oligonucleotide stoppers and one further oligonucleotideprimers. Apart from this difference, the methods comprise the inventivesimilarity to provide limited and well defined amplification productsthat can be amplified in well controlled fashion and most importantly,with a unitary concentration distribution over the length of thetemplate nucleic acid. The inventive amplification products can befurther characterized and used. They are the desired products that areobtained and not discarded, like in template suppression methods. Thepresent invention generates amplified nucleic acid products that betterrepresent the template molecules that need to be analyzed and preparesthose for seamless integration with subsequent analysis methods such asnext generation sequencing or as sequence library. If the stoppers areat the same time primers, it is possible to provide one continuoussequence based on only one template molecule. This continuous sequenceof the amplified product can be provided as single molecule when theindividual product sequences are covalently connected, e.g. ligated.Such connection or ligation reaction can be performed while hybridizedto the template strand to secure that the products are connected in thesame order as the template strand (while of course being the reversecomplement strand).

The advantages of the present invention are most prominent with longtemplate nucleic acids that are amplified according to the invention.Such templates may be at least 100 bases, at least 1000 bases (1kb), atleast 2 kb, at least 4 kb, at least 6 kb, at least 10 kb, at least 20kb, at least 30 kb, at least 40 kb, at least 50 kb, in length.

In case of a reverse transcription an embodiment of the presentinvention relates to a method of reverse transcribing an RNA moleculecomprising hybridizing at least two primers to a template RNA moleculeand extending primers utilizing an RNA dependent DNA polymerase, whereinthe extension product of an upstream primer (downstream in the directionof the template) does not displace the extension product of a downstreamprimer (upstream in the direction of the template). This embodiment ingeneral essentially comprises a primer extension reaction using a firstprimer that is extended but stops at (does not displace) a second primerthat is also extended.

However, for a complete representation of a given template by theelongation product, direct connection of the elongated nucleotides isnot necessarily required. A sample of template nucleic acids usuallycontains many template molecules that have the same sequence. By usingmany, more than one, primer and stopper combinations it is possible tohave a complete representation of such a sequence by the many elongationproducts. Having many short elongation products is often a requirementof a nucleic acid library that fully represents the template. Thus thepresent invention provides the method of preparing a nucleic acidlibrary by providing the elongation products. The library may containthe elongation products in a mixture. Preferably the elongationproducts, especially of templates of the same sequence, containoverlapping sequence portions. Overlapping sequence portions are easierfor complete sequence assembly, as e.g. required in NGS methods and aredesired to provide a library that can be used to clone any specificsequence therein without or with limited interruptions by reaching thesize limits of an individual elongated nucleic acid.

In case of a reverse transcription such an embodiment of the presentinvention relates to a method of reverse transcribing an RNA moleculecomprising hybridizing at least two oligonucleotides, one a primer theother a stopper, to a template RNA molecule and extending the primerutilizing an RNA dependent DNA polymerase, wherein the extension productof the upstream primer (downstream in the direction of the template)stops at (does not displace) the position of the downstream stopper(upstream in the direction of the template). Said stopper is notextended. This embodiment in general essentially comprises a primerextension reaction using a first primer that is extended but stops at(does not displace) an oligonucleotide stopper that is not extended.

It is possible to combine the inventive embodiments, e.g. by using atleast two primers and a stopper, a first upstream primer that isextended downstream, which extension reaction stops at the position of asecond, downstream primer that is also extended downstream, whichextension reaction in turn stops at the position of the oligonucleotidestopper.

In special embodiments no oligonucleotide stoppers that cannot beextended are used. In such embodiments only extendable primers may beused during amplification/transcription.

In other embodiments it is desired to obtain amplification products thathave been stopped at oligonucleotide stoppers. To this end a skilled mancan e.g. select such elongated nucleic acids by well-known methods, suchas by labeling, including immobilization onto a solid phase (e.g. beadsor a solid surface) or by attaching a barcode sequence or sequence tag.The elongated products may also be ligated to the oligonucleotidestopper—similar to the ligation with primers with stopper function inorder to provide one long product molecule as mentioned above. Labelingand ligating to the oligonucleotide stopper can be combined, e.g. bylabeling the oligonucleotide stopper and ligating the extension productto a labeled oligonucleotide stopper. Of course, labeling of theoligonucleotide stopper can be performed after ligation with theextension product. Such labeling allows the easy handling, selectionand/or amplification of the elongated products. E.g. a sequence tag canbe used for further selected amplification of said elongated and solabeled elongation products by using primers that hybridize to such atag and can initiate an amplification reaction, e.g. PCR, of said(previously) elongated nucleic acid product. This is particularlyadvantageous if a multitude of different elongation products areobtained and many are labeled with the same sequence tag. In case ofsequence libraries this method allows an easy amplification of an entirelibrary—still with a consistent representation of the amount of allsequences over the entire length of the original template.

In preferred embodiments more than one oligonucleotide primer is used,which functions similar as the first primer but has a different primersequence, the sequence that anneals to a target template. In particularthe inventive method according to this embodiment further comprisesannealing an additional oligonucleotide primer to said template nucleicacid and elongating said additional oligonucleotide primer until theelongating product nucleic acid reaches the position of anotheroligonucleotide primer or an oligonucleotide stopper. In especiallypreferred embodiments at least 1, at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 30, at least 40, at least 50 or moredifferent oligonucleotide primers are used. “Different oligonucleotideprimers” is understood that they differ in a primer sequence but, ofcourse, may share other similar sequence portions, such as sequencetags. Such sequence tags are preferably used in a further amplificationreaction of the elongated products by using primers that anneal to thesequence tags. Thus, with one primer all potentially different productscan be amplified. In a special embodiment the oligonucleotide primersare random primers. “Random primers” is to be understood as a mixture ofdifferent primers with different primer sequence portions, with a highvariance due to a random synthesis of at least a portion of the primersequence. Random primers potentially cover the entire combinatory areafor said sequence. The random sequence primer portion of the randomprimer may cover 1, 2, 3, 4, 5, 6, 7, 8 or more nucleotides which arerandomly selected from A, G, C or T (U). In terms of hybridizingsequences of primer sequences T and U are used interchangeably herein.The full combinatory possible area for a random sequence portion is mn,wherein m is the number of nucleotide types used (preferably all four ofA, G, C, T(U) and n is the number of the random nucleotides. Therefore arandom hexamer, wherein each possible sequence is represented, consistsof 4⁶=4096 different sequences.

Likewise it is also possible to use more than one oligonucleotidestopper in any one of the inventive methods. Said additional stoppersact similar as the first stopper, but differ in the sequence aligned tothe template. The same as described above for additional primers appliesfor the additional stopper—of course with the difference that thestoppers are not suitable for elongation reactions. Therefore, forconsistency the region of the oligonucleotide stopper that hybridizes tothe template is also referred to as “primer sequence”. Said primersequence may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22 or more nucleotides long. The invention thus provides amethod as defined above further comprising annealing an additionaloligonucleotide stopper to said template nucleic acid and wherein insaid elongation reaction said additional oligonucleotide stopper is notelongated. In preferred embodiments at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 15, at least 20, at least 30, at least 40, atleast 50 or more different oligonucleotide stoppers are used. Theoligonucleotide stoppers may be random oligonucleotide stoppers,comprising a random primer sequence that anneals to the template. Asdescribed above for oligonucleotide primers, also oligonucleotidestoppers may share similar sequence portions, such as tags or barcodesthat may be used for amplification or identification of the products.Such kinds of labels allow easy identification in a sequence librarycomprising the inventive amplification products.

As described in the introduction a (randomly) primed cDNA librarywithout control of the elongation reaction (and its stop) distorts theactual RNA representation of sequence portions. Even on e.g. short mRNAtemplates (200-1000 nt) multiple priming events can occur and with thestrand displacement the 5′ side of RNA molecules will be present in morecopies than the 3′ side (see also FIG. 1). Therefore when measuring theconcentration of a certain gene transcript different values will beobtained when probing for sequence portions at the 5′ or 3′ end. Shortrandom probes are commonly used in microarray and quantitative PCRanalysis. This leads to severe distortions of the concentrationmeasured. Furthermore, this distortion will be greater when comparinglong and short transcripts, as the 5′ ends of high abundant longtranscripts will be even higher represented than the 5′ ends of shortertranscripts. When globally analyzing the concentration of transcriptssuch as in high throughput sequencing (e.g. next generation sequencing)in addition to distorting concentrations detecting rare shorttranscripts becomes even less likely than detecting rare longtranscripts. As the differential expression of gene transcripts andtheir splice variants is an important part of phenotype analysis, it isimportant that each sequence portion of a transcript is represented inthe generated cDNA library at the correct abundance.

These problems are solved by the present invention. Applying theinventive methods to provide well defined transcripts, stopped at aprimer or stopper position (preferably with inhibited stranddisplacement of the reverse transcriptase) during the generation of cDNAfrom a multitude of RNA molecules as for instance in the generation of acDNA library of mRNA molecules, enables the equal representation of eachportion of the RNA molecules. For instance mRNA can be primed withrandom primers such as random hexamers that are modified to ensure thatthe random primer does not get displaced by the reverse transcriptase.Any random primer can be used that can start reverse transcription frommultiple sites from the template RNA molecules.

The present invention also provides for methods that enable the covalentjoining (e.g.: through a ligation) of the obtained elongated products asthey are provided in a hybrid with the template (see also FIG. 2c ).This enables the generation of a full length amplified nucleic acidmolecule.

Using primers that provide for a 5′ phosphate, it has been found thatthe “short” elongated nucleic acids—that are essentially fragments ofthe complementary strand to the template strand—can be ligated whilestill being hybridized to the template. This will result in long and inmost cases full length amplified nucleic acid molecules that are adirect representation of the template. Being able to preserve theinformation of the full length sequence of a template is, for instance,important when splice variants of a gene need to be analyzed. Especiallywhen splicing of multi exonic genes is complex the analysis of singlesplice junctions alone will not yield unambiguous information towardsthe full length sequence of the transcript variant involved. However,only oligo dT priming in case of mRNA or priming from a 3′ ligateduniversal linker from any nucleic acid template will lead to anunderrepresentation of the 5′ side of the template. In other words, thelonger the template is the less likely it becomes that the molecule willbe reverse transcribed to full length. As can be seen in FIG. 15, when astandard oligo dT primed RT is used on average after 6 kb only ˜10% ofRNA molecules are reverse transcribed to full length. As the presentinvention provides—in one embodiment—for methods that will start thepolymerization from two or more positions of a template molecule with anelongation reaction that is stopped at the position of a downstreamprimer, the extension product of the upstream primer can be ligated tothe 5′ end of the downstream primer. By covalently joining bothextension products when in hybrid with the template a longer sequence iscreated. In continuation when many different primers are usedpotentially all templates present in the sample can be transcribed andby ligating the short extension products a full length amplified copy ofthe template can be created (see also FIG. 2c, d ).

Likewise, according to the inventive embodiments utilizingoligonucleotide stoppers, a ligation with the elongated product can beperformed to obtain well defined amplified nucleic acids of anamplification of a sequence between the primer and the stopper.

In preferred embodiments the oligonucleotide primers and/oroligonucleotide stoppers are phosphorylated or adenylated on the 5′ end.This measure helps to easily ligate the oligonucleotide primers orstoppers to another nucleic acid, such as the elongation product, in oneor few steps using a ligase. In some embodiments, especially whenmixtures of many primers and stoppers are used, it is possible to onlyprovide the stoppers with such a modification to ensure ligation ofelongated nucleic acids with stoppers and prevent ligation with otherprimers.

Any ligase known in the art can be used, such as T4 RNA ligase, T4 DNAligase, T4 RNA ligase 2, Taq DNA ligase and E. coli ligase.

To prevent any non-hybridized primers or stoppers from being ligated apreferred embodiment of the invention uses a double strand specificligase such as T4 RNA ligase 2 or T4 DNA ligase. Double stranded DNA isthe natural substrate of T4 DNA ligase and DNA-RNA hybrids are poorsubstrates [29]. In order to overcome this inefficiency Mg²⁺ can bereplaced with Mn²⁺ as a divalent ion for the enzyme [30]. In oneembodiment of this invention the addition of PEG to the ligationreaction is shown to increase the ligation efficiency of DNA moleculesin an RNA hybrid even in a Mg²⁺ containing ligase buffer.

Optionally a T4 RNA ligase, which is deficient of adenylation (e.g.truncated), can be used for ligation which relies on the presence ofadenylated DNA fragments that are in the hybrid with the RNA(adenylation with T4 DNA ligase occurs exclusively in double strandednucleic acids). It can be added additionally to the ligation reaction tofurther increase the efficiency. The ligation happens exclusively in thehybrid and was previously considered very inefficient form of ligation[31]. In U.S. Pat. No. 6,368,801 a ligation of 2 DNA molecules (withribonucleotides at their 3′ and 5′ ends) in an RNA hybrid by T4 RNAligase is described. However T4 RNA ligase is not specific for hybridssince it ligates all single stranded nucleic acids containingdeoxyribonucleotides at their 5′ or 3′ end (RNA to DNA, RNA to RNA, DNAto DNA providing there is one deoxyribonucleotide at the 3′ and 5′ end)as well. The present invention includes the addition of PEG in theligation reaction, which allows the ligation to take place in the RNAhybrid. PEG has been used in single stranded ligation reactions as amolecular crowding agent, increasing the likelihood of the donoroligonucleotide ligase complex interacting with the acceptor (3′OH) bydecreasing the effective reactive volume [32]. Here in contrast thedonor oligonucleotide ligase complex is already next to the acceptor andPEG serves to change the conformation of the RNA:DNA hybrid bycondensing the double helix into a conformation that is more reminiscentof a DNA:DNA helix, so that the T4 DNA ligase that normally is specificfor ligating two DNA molecules in a hybrid with a DNA strand, can ligatetwo DNA molecules that are in a hybrid with an RNA molecule.Alternatively T4 RNA ligase 2, which is a double strand-specific ligasecan be used.

Other additives such as Tween-20, NP-40 could be added additionally orinstead of PEG for efficient ligation in the RNA hybrid. Within thescope of the invention the ligation reaction requires 12%-25% finalPEG-8000 (v/v). A variety of PEG molecular weights and compounds can beused, and the skilled experimenter will appreciate that the identity andconcentration of the additive can be varied to optimize results. In thecontext of the present invention, an effective amount of PEG is anamount sufficient to permit ligation activity in an RNA hybrid. In a 20μl RT reaction the optimal PEG concentration for a ligation in an RNAhybrid was found to be 20%. However, it is apparent to the skilled inthe art that optionally the reaction volume and the PEG concentrationcan be increased or decreased (e.g. descrease in volume, increase in PEGamount or concentration) to potentially further optimize the ligationefficiency of 2 DNA molecules in an RNA hybrid by T4 DNA ligase or T4RNA ligase 2. Other additives such as 1 mM HCC and/or pyrophosphatasecan further increase the ligation efficiency in an RNA hybrid.

Pre-adenylated oligonucleotides can be inserted into the reaction andused with truncated T4 RNA ligase, provided that any unhybridizedoligonucleotides have been removed prior to the ligation reaction. Asmentioned before in another embodiment of this invention truncated T4RNA ligase 2 can be added in addition to T4 DNA ligase to further boostthe ligation of DNA fragments in an RNA hybrid.

Therefore it is preferred that the ligase is a double strand specificligase such as T4 DNA ligase or T4 RNA ligase 2 and wherein it ispreferred that Polyethylene glycol is used at a concentration between12% and 25%.

Apart from a representative template amplificate synthesis an additionalbenefit of the present invention is the improved efficiency ofgenerating long product nucleic acids, especially cDNA synthesis,resulting in higher sensitivity of detection and longer products (seeExample 5 and 6).

In preferred embodiments any one of the oligonucleotide primers maycomprise a sequence tag. Such a sequence tag is a label in form of aunique pre-selected nucleic acid sequence that can be used to detect,recognize or amplify a sequence labelled with said tag. Preferably auniform sequence tag is attached to more than one of the oligonucleotideprimers, especially preferred to all of the oligonucleotide primers.Such a sequence tag is preferably prevented from annealing to thetemplate, e.g. by being hybridized to a complementary nucleic acid.Sequence tags can be attached to the 5′ end of the oligonucleotidestopper—so as not to prevent the elongation reaction of the primer atits 3′ end.

In preferred embodiments any one of the oligonucleotide stoppers maycomprise a sequence tag. Such a sequence tag is a label in form of aunique pre-selected nucleic acid sequence that can be used to detect,recognize or amplify a sequence labelled with said tag. Preferably auniform sequence tag is attached to more than one of the oligonucleotidestoppers, especially preferred to all of the oligonucleotide stoppers.Such a sequence tag is preferably prevented from annealing to thetemplate, e.g. by being hybridized to a complementary nucleic acid.Sequence tag can be attached to the 3′ end of the oligonucleotidestopper. At this position the tag does not hinder the contact of theelongating product nucleic acid to the 5′ end of the stopper—resultingin well-defined products. It also allows ligation of the elongatedproduct to the oligonucleotide stopper. Alternatively the tag may be onthe 5′ end of said stopper, however, prevented from hybridization to thetemplate so that the elongation reaction still reaches the 5′ end of theprimer region of the stopper. Said tag preferably comprises a free 5′end so that the elongated product can be ligated to the tag forlabelling of the elongated product by said tag. A free 5′ end of the tagthat can be easily ligated to the product is preferably provided in thevicinity of the 3′ end of the elongated product. This can be achieved bye.g. providing the tag hybridized to a complementary region of theoligonucleotide stopper, said complementary region being hybridized withthe tag and attached to the 5′ end of the primer region of theoligonucleotide stopper (for an example see FIG. 4).

The inventive labelling step of the elongated product when it reachesthe position of the oligonucleotide stopper (or another primer),preferably a stopper, can be achieved with any known means readilyavailable in the art. Such means include attaching a chromophore,fluorophore or simple phase separation by binding to a solid phase andwashing of said solid phase to remove all non-bound nucleic acids,thereby isolating the labelled nucleic acid. In preferred embodimentssaid labeling step comprises ligation with a sequence tag. A sequencetag may be attached to said oligonucleotide primers or oligonucleotidestoppers. Labelling may also comprise ligation with said oligonucleotideprimers or oligonucleotide stoppers, which comprise a sequence tag.

For many downstream analyses it is preferred that oligonucleotides, suchas primers, stoppers, blockers or linkers get depleted. Especially, whenan amplification of the library utilizing the universal linker sequencesis necessary or desired it is preferable to deplete the un-ligatedlinkers.

This can be achieved through for instance a size exclusion, retainingthe oligonucleotides (shorter) in an appropriate bed, and recovering thelibrary (longer). Another possibility is to bind the longer library to asilica based carrier while not retaining the shorter oligos. Suchdiscrimination can also be carried out to distinguish between the singlestrandedness of the oligo and the double strandedness of the librarywhen still in hybrid with the template. Another possibility for lengthbased purification are methods based on PEG precipitation. The higherthe PEG concentration the shorter the nucleic acids that can beprecipitated. For instance, it was found that using a 12.5% PEGprecipitation, all small fragments (below 60 nts) will stay in thesolution and only the cDNA and the linker ligated library willprecipitate.

In a preferred embodiment a beads-based clean-up approach is used. OligodT coupled beads or Streptavidin beads to isolate biotin labeled oligodTprimers are commercially available. A beads based clean up has theadditional advantage that both RT and ligation can be performed on thebeads. After hybridizing the mRNA to the oligo dT (biotin tagged or onbeads) and to the starter and stoppers, any non-hybridized starter andstopper excess can be washed off before starting the RT reaction. Thusin preferred embodiments of the invention the template nucleic acid isimmobilized on a solid phase or solid support, preferably beads. Theamplified nucleic acids hybridized to said template nucleic acid maythen be washed.

Another embodiment of the invention is that RT and ligation can beperformed in one reaction step, simply by adding Ligase (e.g.: T4 DNAligase or T4 RNA ligase 2), 10% PEG, and 0.4 μM ATP to the reversetranscription reaction in a regular reverse transcription reactionbuffer. Although 30 min incubation can be used, a better yield wasobtained using 2 h incubation at 37° C. Following another washing step(4 washes) the RNA can then be hydrolyzed to obtain the di-tagged cDNAlibrary, which can then be inserted into a PCR reaction. RT and ligationcan also be performed in two successive reaction steps, re-buffering thereaction simply by washing the beads. However, in a preferred embodimentof the invention a simultaneous RT/ligation reaction is performed sincethis resulted in similar yield as the two step protocol, but has theadvantage of reduced hands on and incubation times. Simultaneous RT andligation can be performed by addition of a DNA polymerase and ligase inone reaction mixture with the template nucleic acid.

In preferred methods of the invention said elongated products areamplified. Amplification may comprise using tag specific primers toamplify elongated products that comprise a 5′ and/or 3′ tag, stemmingfrom the tag-labelled oligonucleotide primer and/or stopper,respectively. Amplification is preferably by PCR. Sequence tags suitablefor primer hybridization in a following amplification cycle are alsoreferred to herein as “linkers”.

Currently any preparation of small cDNA fragments for high throughputsequencing such as NGS involves fragmentation of RNA and in most cases amultistep procedure to introduce the 5′ and 3′ linker tags foramplification and bar-coding. For instance Epicentre's ScriptSeq™mRNA-Seq Library Preparation Kit uses terminal-tagging technology (US2009/0227009 A1) and random-priming on chemically fragmented RNA(depleted of ribosomal RNA). However, any fragmentation of mRNA(chemical or physical) introduces an un-defined and not foreseeablebias. In addition during fragmentation protocols a portion of the RNA isdegraded or gets lost. Therefore the present invention is ideally suitedto generate cDNA libraries of a rather defined short size without theneed of RNA fragmentation.

In addition when the RNA is fragmented many additional 5′ ends arecreated. Reverse transcriptases have the tendency to add a fewnon-template nucleotides when they reach the 5′ end of the template RNAand use these nucleotides for priming second strand synthesis. Thereforewhen RNA is fragmented more 5′ ends are generated and therefore moresecond strand synthesis is initiated. One important question during RNAsequencing is the question from which DNA strand an RNA was transcribed.Especially in the analysis of sense and antisense transcription a highstrandedness (conservation of strand information) of the librarysequenced is required. Therefore as no RNA digestion is needed in thepresent invention a much higher degree of strandedness can be achievedin the cDNA library generated (see also example 9) compared to librarypreparation methods that include RNA fragmentation.

Of course this method can be employed for the generation of fragments toany kind of template nucleic acid and is not limited to RNA. Suchfragments are provided by the inventive elongated products or amplifiednucleic acid portions. The template nucleotide sequence could also beDNA. Hence a library preparation using the techniques described in thisinvention can also be started from genomic DNA or PCR products. Sincereverse transcriptases are also accepting DNA templates [33], all stepscan basically be performed as described within this invention.Optionally also DNA dependent polymerases can be used if DNA is used astemplate.

As for many analysis methods such as NGS, it is preferable to havedefined universal linker sequences present on the 3′ and/or 5′ end ofthe cDNA. Such linker sequences can for instance serve as priming sitesfor PCR amplification to enrich for a library or priming sites forbridge amplification on a solid surface or to prime a sequencingreaction. Therefore it is within the scope of the invention that linkersequences are ligated to the amplified nucleic acid portions. However itis preferred that the 5′ linker sequence is directly introduced with theprimers (see also FIG. 3 (L1)). Here the 5′ linker sequence is a 5′extension to the sequence that is used for priming the polymerase.

Alternatively or in addition a linker sequence can be introduced on the3′ end of the elongated nucleic acid product, e.g. by using a stopperoligo (FIG. 3. (S1, Sm)) that has a linker sequence added on its 3′ end(FIG. 3. (L2)). The 5′ end of the stopper oligo can be ligated to the 3′end of the extension product (FIG. 3b ). The specific ligation reactionensures that the 5′ end of the stopper oligo is not strand displaced.Therefore in a preferred embodiment a stopper oligonucleotide is addedto the reverse transcription reaction and wherein the stopperoligonucleotide has a 3′ linker sequence extension.

In an alternative version—as illustrated in FIGS. 5 and 8—the starterand stopper oligonucleotide are at least partially hybridized to eachother. The 5′ phosphorylated stopper oligonucleotide can either stillhybridize to the template strand (as shown in FIGS. 8a -d,f-h) or nothave any hybridization to the template strand (see FIG. 8e ). In thiscase the extended oligo referred to as a starter has to be the actualoligo stopping the strand displacement and hence contains preferablymodifications that stop strand displacement. The extended strand from amore upstream starter will be stopped at the next starter and thephosphorylated oligo previously referred to as the stopper that ishybridized to the starter will be ligated to the extended cDNA strand.

In 8e an even more elaborate starter/stopper combination is shown. Thedetails on the linker sequence L1 and L2 can be found in the descriptionof FIG. 9.

Finally the sequence tag (“linker”) of the starter and stopper oligo canbe joined (see FIG. 8h ) in order to introduce sequence tags into thecDNA, but keep the sequence of the individual extension products inorder as they are now covalently linked to each other through theirstarter stopper sequence tags.

It is preferred that a polymerase is used during elongation that has lowor no terminal transferase activity, as to not add non templatednucleotides to the 3′ end of the extension product upon reaching theposition of the 5′ end of the stopper oligo, as a 3′ overhang wouldreduce the specificity and efficiency of the ligation reaction. Reversetranscriptases with low terminal transferase activity are for instanceSuperscript III (Invitrogen); RTs with no terminal transferease activityare e.g. AMV-RT.

Alternatively or in addition terminal transferase activity and hencealso second strand synthesis can be inhibited by the addition ofActinomycin D. Actinomycin D can be added to the polymerisation reactionin sufficient amounts to avoid second strand synthesis and/or to reducestrand displacement of the polymerase as compared without actinomycinaddition. In a preferred embodiment Actinomycin D is added to the RTreaction at a final concentration of about 50 μg/ml, also higher orlower concentrations can also be used, such as e.g. 5 μg/ml to 200μg/ml.

In addition or alternatively an overhang can be digested by a singlestrand specific nuclease, preferably a 3′-5′ exonuclease, such as butnot limited to Exonuclease I (3′-5′ ssDNA digestion), Exo T5 (3′-5′ ssor dsDNA digestion).

One of the goals of the invention is to control for any bias introducedinto the sequence library obtained by the amplified nucleic acidproducts, and this means in most cases to minimize bias. Linkersequences that are introduced as an extension to random or semi-randompriming sequences can also participate in the hybridization to thetemplate. This will add a bias to the library generated. It is thereforepreferred that at least the nucleotides of the linker that lay next tothe primer or the stopper sequence are inhibited from participating inthe hybridization to the template. This can be achieved throughdifferent means. For instance an oligonucleotide with a reversecomplement sequence to the linker sequence can be added to the reaction(see also FIGS. 5c-f ; FIGS. 6c-f ). In that case the reverse complementwill compete with the template for hybridization to the linker sequenceand by using excess of reverse complement the participation of thelinker sequence in priming the RT or hybridizing the stop oligo can beeffectively quenched.

It is preferred to provide the reverse complement with nucleotidemodifications that enhance the stability of linker:reverse complementhybrid, by using for instance LNAs. However, any other modification canbe used that enhances the binding energy (see also FIG. 6e ; FIG. 7e ).

Furthermore, it is preferred that the primer and/or stopper are added asa premade adapter to the reverse transcription. This means thatessentially all linker sequences are in a hybrid with their reversecomplement strands and so the linker sequence is inhibited fromparticipation in the hybridization reaction.

Therefore, in a preferred embodiment a reverse complement sequence tothe linker sequence part of the oligonucleotides is added to thereaction, preferably already in a hybrid with the oligonucleotide primerand it is further preferred that the Tm of the reverse complement oligois raised by e.g. introducing modifications such as LNAs, 2′fluoronucleotides or PNAs.

In a preferred embodiment the reverse complement sequence is covalentlylinked to the linker sequence (see FIG. 6g ; 7 g). This can be either indirect continuation to the linker sequence or through a nucleotidehairpin or spacers such as C3, C6, C12, or any other moiety. In additionthis moiety can be a modification that enhances adapter formation. Asdescribed before it is preferred that the nucleotides of the reversecomplement are modified to enhance hybridization to the linker sequence.Therefore it is preferred that the reverse complement to the linkersequence is either directly connected to the linker sequence or througha nucleotide hairpin or spacer such as C3, C6, C12 or any other moiety.

In a most preferred embodiment a sequence tag on the primer (L1) and asequence tag on the stopper (L2) comprise at least partiallycomplementary sequences which allow starter and stopper to form hybrids(see FIGS. 5 and 8). In that manner the sequence tag (“linker”)sequences will not hybridize to the template strand and as an addedbenefit a stopper-ligation will happen in immediate proximity to thenext starting event, minimizing any gaps between starting and stoppingevents. Thus; in preferred embodiments of the invention at least one ormore or all oligonucleotide stopper(s) is/are hybridized to at least onefurther oligonucleotide primer. In an alternative embodiment oradditional embodiment in combination, at least one or more, preferablyall, oligonucleotide primer(s) is/are hybridized to an oligonucleotidestopper. Especially preferred, any one of the oligonucleotide stoppersand any one of the oligonucleotide primers comprises a sequence tageach, and preferably wherein the sequence tag of the oligonucleotideprimers is at least partially complementary with the sequence tag of theoligonucleotide stopper thereby enabling hybridization of theoligonucleotide stoppers and the oligonucleotide primers with each otherat least in a part of the respective sequence tag.

As any free 3′ OH can potentially serve as an acceptor duringpolymerization or ligation, it is preferred that any free 3′ OH (exceptthe one on the oligonucleotide primer) is blocked (see also FIG. 6f ;FIG. 7f ). Many blocking groups are known in the art. Provided forreference but not limiting are dideoxynucleotides, C-spacers andphosphate groups. In addition, the 3′ end of the reverse complement inthe priming adapter can also be provided with an overhang (see FIG. 6d). Correspondingly, the linker sequence of the stopping adapter can beprovided with a 3′ overhang (see FIG. 7d ). Therefore it is preferredthat the 3′0H of the oligonucleotides that do not participate in theprimer extension reaction are blocked and/or where provided in a hybridthat the 3′ end has an overhang over the 5′ end.

When introducing the linker sequences together with the primer and/orthe stopper oligonucleotides the sequence between the linker sequencesreflects the template RNA sequence. As a mis-hybridization of the primeror the stopper to the template is much more likely than theincorporation of a wrong nucleotide during polymerization, the primersequence and/or the stopper sequence are more likely to contain an errorthan the polymerized sequence. Therefore, when e.g. sequencing thelibrary in an NGS experiment, it would be preferable that the linkersequence that contains the sequencing primer is next to the primerextension product. A solution to this problem is shown in FIG. 4. Herethe L2 sequence is in a hybrid with its reverse complement that has beenintroduced at the 5′ side of the stopper oligonucleotide. In this mannerthe L2 sequence can be ligated to the primer extension product once thepolymerase reaches and stops at the stopping oligo. However, thesequence of the stopping sequence of the oligo is not included into thelibrary. Therefore, starting any sequencing reaction from the L2sequence will not include a potentially mis-hybridized stopper sequence.Therefore, it is preferred that the linker sequence is on the 5′ end ofthe stopping oligonucleotide and the 5′ end of a reverse complement tothe linker sequence is ligated to the 3′ end of a strand displacementstopped extension product as a sequence tag. The same principle appliesfor the starter-“stopper” combination shown in FIG. 8e with the soledifference that the oligo responsible for stopping is actually thestarter of the next library fragment.

Thus in preferred embodiments the oligonucleotide primer oroligonucleotide stopper is hybridized with a sequence tag asoligonucleotide label. Especially preferred said sequence tag ispreferably hybridized to a portion on the 5′ end of said oligonucleotideprimer or oligonucleotide stopper. The next, e.g. 1, 2, 3, 4 ,5, 6 ormore, nucleotides of said primer or stopper in 3′ direction next to thenucleotides of said primer or stopper that are hybridized to saidsequence tag are hybridized to the template. This allows positioning ofthe 5′ end of the tag near the 3′ end of an elongation product ofanother primer, that is located upstream of the oligonucleotides primeror oligonucleotide stopper with the hybridized sequence tag so that theelongated 3′ end of said further primer can be ligated to the 5′ end ofsaid sequence tag. Such tags may also be used for subsequentamplifications reactions of the ligated products and are also referredto as “linkers” herein.In preferred embodiments the oligonucleotide primer and/oroligonucleotide stopper comprises a nucleotide modification increasingthe Tm or stiffening the sugar phosphate backbone of saidoligonucleotide. These modifications to increase the Tm are tostrengthen hybridization to the template to secure stopping of theelongation reaction and prevent displacement of the primer or stopper.Such modifications are known in the art from oligonucleotide blockers,such as described in GB 2293238, US 2002/0076767 A1, U.S. Pat. No.6,391,592 B1, WO 99/61661, WO 02/086155, U.S. Pat. No. 5,849,497, WO2009/019008. Suitable modifications include one or more of themodifications selected from 2′fluoro nucleosides, LNA (locked nucleicacid), ZNA (zip nucleic acids), PNA (Peptide Nucleic Acid). Further theTm can be increased by using intercalators or additives thatspecifically bind to nucleic acids, such as Ethidiumbromid, Sybr Green.Preferred intercalators are specific for RNA:DNA hybrids. The number ofmodified nucleotides may vary, depending on other measures taken toincrease the Tm. Preferably 1, 2, 3, 4, 5 or 6 nucleotides are modified.Preferably the modified nucleic acids are on the 5′ side of the primersequence portion, the portion of the oligonucleotide primer or stopperthat can hybridize or anneal to the template. Preferably 1, 2 or 3 5′nucleotides are modified. DNA polymerases may have an intrinsic stranddisplacement activity, especially reverse transcriptases to denaturesecondary RNA structures. A polymerase having nucleotide stranddisplacement activity may be used for the elongation.

As DNA polymerases, especially reverse transcriptases, can displace aDNA oligonucleotide from a template strand of RNA at least as good asdissolving secondary or tertiary structure, the hybridization of theoligonucleotide has to be enhanced in order to stop strand displacementof the reverse transcriptase. This can be achieved by usingmodifications to the oligonucleotide itself or by using additives thateither stabilize the hybridization of the oligonucleotide or that stopthe reverse transcriptase. Modifications to the oligonucleotides thatreduce or inhibit the strand displacement activity of the reversetranscriptase are for instance 2′ fluoro nucleosides [15], PNAs [16, seeFIG. 2; 17], ZNAs [18, 19], G-Clamps (U.S. Pat. No. 6,335,439, acytosine analogue capable of Clamp Binding to Guanine) or LNAs (US2003/0092905; U.S. Pat. No. 7,084,125) [20, 21]. These modifications ingeneral increase the melting temperature of the oligonucleotide, byincreasing the local hybridization energy of the oligonucleotide to thetemplate RNA strand. Some also stiffen the sugar phosphate backbonefurther inhibiting strand displacement by the reverse transcriptase.

Alternatively or in addition, the hybridization of the oligo to the RNAtemplate can be altered by using different additives that bind orintercalate to the nucleic acids. For instance, ethidiumbromide,SybrGreen (U.S. Pat. No. 5,436,134; U.S. Pat. No. 5,658,751; U.S. Pat.No. 6,569,627) or acricidine can be used. Other compounds that can bindto dsNA are actinomycin D and analogues [22]. However they potentiallyalso stabilize RNA secondary structure.

Therefore, it is preferred that such intercalators or additivesspecifically bind to RNA:DNA hybrids. Examples are aminoglycosides ofthe Neomycin family (Neomycin, Ribostamycin, Paromomycin and Framycetin[23]). Additives that alter the hybridization properties of theoligonucleotide can also be covalently included into the oligonucleotidestructure [23].

The hybridization energy and kinetics can be changed to inhibit thestrand displacement by the reverse transcriptase by the addition ofnucleic acid binding proteins such as single stranded binding proteinsuch as TtH SSB [24] or Tth RecA [25].

It will be apparent to those skilled in the art that those additives arejust examples and any other compound, base modification or enzymeleading to an increased hybridization of the oligonucleotide to RNA canbe used to increase the Tm and hence inhibit strand displacement.

The increase in the Tm should be strong enough to prevent a displacementof any one of the 5′ end nucleotides of the primer region annealed tothe template by an elongating polymerase. In particular, the inventiveTm increase prevents displacement of the 3^(rd), 2^(nd) and/or 1^(st)nucleotide downstream to the 5′ end of the primer region.

In certain embodiments of the invention the strand displacement needs tobe stopped right at the first 5′ nucleotide of the downstream primer,especially when the cDNA fragments are ligated to each other or to alinker as described below. The strand displacement of an oligonucleotideis reduced or inhibited by using nucleotide modifications that increasethe Tm or stiffen the sugar phosphate backbone of oligonucleotide, bye.g. including 2′ fluoro nucleosides LNAs, PNAs, ZNAs or PNAs or byusing intercallators or additives that specifically bind to nucleicacids such as Ethidiumbromid, Sybr gold, SybrGreen, preferablyintercalators that are specific for RNA:DNA hybrids.

Therefore it is preferred that the binding of the oligonucleotideprimers are specifically Tm enhanced at their 5′ ends to prevent theelongating polymerase from displacing them. Such modifications includebut are not limited to LNAs, PNAs, ZNAs, acridine or fluorophores.

Oligonucleotides with an increased Tm at their 5′ end such asLNA-modified oligonucleotides enable a stop right at the start of thenext primer. It is within the scope of the invention to combine thestrand displacement stop by using the LNA-modified oligos together withthe displacement synthesis deficient mutants such as Y64A M-MLV or F61WHIV or any other reverse transcriptase with impaired displacementsynthesis as well as lowering the reaction temperature and usingdifferent additives to increase the binding of oligonucleotides to theRNA.

Preferably C and/or G nucleotides are modified. Even unmodified thesenucleotides have a higher Tm than A or T due to increased hydrogenbridge formation when complementary annealed. In preferred embodimentsthe oligonucleotide primer and/or oligonucleotide stopper comprises atleast one, at least 2, at least 3, at least 4, at least 5, at least 6modified nucleotides being selected from G or C. These modifiednucleotides are preferably at the 5′ end of the primer sequence asmentioned above.

Most efficient strand displacement stop is achieved by G or C bases asthey increase the local Tm of the primer or stopper. Hence semi-randomprimers or stoppers (hexamers, heptamers, octamers, nonamers, etc.)containing at least two, more preferably three or more Gs or Cs or acombination of Gs and Cs. It is most preferred if these Gs or Cs aremodified to increase the local melting temperature, as is the case whenusing LNA modified bases. It is most preferred that at least 1, at least2 or at least 3 LNA modified bases are used at the 5′ end of the primer.Therefore, it is preferred that at least two, at least 3 modifiednucleotides are used optionally chosen from G or C.

Several methods and means exist to ensure that the elongation reactionis stopped when the elongation reaction reaches the position of anoligonucleotide stopper or further or additional primer annealed to thetemplate. This stopping is also referred to as a prevention of stranddisplacement herein. Strand displacement is a particular problem forreverse transcription due to increased or high strand displacementactivities of reverse transcriptases. The inventive step of inhibitingthe reverse transcriptase to strand displace the cDNA of an alreadycopied RNA portion ensures that any portion of an RNA molecule thatalready got copied is not copied again. Therefore, no copied portion ofthe RNA gets overrepresented in the cDNA library synthesized. Thisinhibition of strand displacement can be achieved through differentmeans, such as decreasing the reaction temperature, using a reversetranscriptase without strand displacement activity, increasing themelting temperature or the hybridization energy of the primer:RNA hybridor increasing the rigidity of the RNA or primer or stabilizing thehelix. In practice, usually a combination of these means is selected toachieve optimal reaction conditions without strand displacement. Aperson skilled in the art is well enabled to select suitable parametersas described herein or known in the art to suit a particular templateand reaction conditions.

One option is to modify the reaction temperature. In general, a reactiontemperature above 37° C. is favored during RT for better dissolvingsecondary structures in the RNA template that leads to a more efficientdisplacement synthesis. In one embodiment stopping of stranddisplacement of the primer is achieved by decreasing the reactiontemperature. Reaction temperatures below 37° C. and down to 4° C. areused to reduce strand displacement. It is preferred that thepolymerization during RT is carried out between 20° C. to 37° C.However, even at these low reaction temperatures the strand displacementstop will not be complete when reverse transcripases are used that havestrand displacement activity and/or a simple stopper oligonucleotide isused that has no modifications that alter its melting temperature.

In one embodiment instead of or in addition to decreasing the reactiontemperature to achieve a better stop of the elongation at said positionof a further primer or a stopper (and reduce strand displacement)reverse transcriptases that are deficient in strand displacement such asthe Y64A M-MLV mutant [12] or the F61W (Phe-61-Trp) HIV mutant [13, 14]can be used. Strand displacement deficient mutants are able to displacethe next primer or stopper for up to 3nts when unmodified. It is withinthe scope of the invention to combine the strand displacement stop bydecreasing the reaction temperature with the usage of displacementsynthesis deficient mutants such as Y64A M-MLV or F61W HIV or any otherreverse transcriptase with impaired displacement synthesis.

A drawback of decreasing the reaction temperature during the RT or usinga reverse transcriptase that has a reduced or impaired stranddisplacement activity is that the reverse transcriptase will havedifficulties reading through regions of RNA secondary structure. Themore stable the secondary structure is the less likely it is that thisportion of the RNA gets copied into cDNA. This means that though no partof one RNA molecule gets copied more than once parts of the RNA thatform a secondary structure will not be copied. This means that someparts of the RNA will be underrepresented in the cDNA library produced.

Therefore, in a preferred embodiment a reverse transcriptase is usedthat has strand displacement activity and/or the reverse transcriptionis carried out at elevated temperatures that sufficiently dissolvesecondary RNA structure. Under these conditions every portion of the RNAis accessible to the reverse transcriptase. However as RNA:RNA hybridsare generally more stable than RNA:DNA hybrids, also the cDNA copy canbe again strand displaced if no further modifications are used.Therefore, in preferred embodiments the reverse transcription is carriedout under conditions that do not allow for secondary or tertiarystructure formation of the RNA template (RNA:RNA hybrids) or underconditions that allow for these secondary structures to be stranddisplaced by the reverse transcriptase, while at the same time theprimer extension product (cDNA copy) cannot be displaced.

Increasing the concentration of monovalent counter-ions also willstabilize the hybrid (but also the RNA secondary structure), as it hasbeen reported for HIV-RT that at 75 mM KCl the strand displacementactivity is impaired though not inhibited [26, 27]. In a preferredembodiment the concentration of monovalent positive ions is preferablyselected from at least 20 mM, at least 30 mM, at least 40 mM, at least50 mM, at least 60 mM, at least 70 mM. Similar concentrations can beindependently selected for single negatively charged ions.

The reverse transcriptase used during the elongation reaction may be aviral reverse transcriptase, and may be selected from the groupconsisting of AMV RT (and mutants thereof such as Thermoscript RT), MMLVRT (and mutants thereof including but not limited to Superscript I, IIor III, Maxima RT, RevertAid, RevertAid Premium, Omniscript, GoScript),HIV RT, RSV RT, EIAV RT, RAV2 RT, Tth DNA polymerase, C.hydrogenoformans DNA polymerase, Avian Sarcoma Leukosis Virus (ASLV) andRNase H—mutants thereof. Mixtures of any of these reverse transcriptasesmay be used. In particular, mixtures of viral RT enzymes may be used,such as mixtures of MMLV and ASLV, and/or their RNase H reduced or RNaseH minus analogs may be used. In any of these methods and compositions,two or more reverse transcriptases may be used, including any reversetranscriptase as described above.

It is within the scope of the invention to combine the stranddisplacement stop by decreasing the reaction temperature with the usageof displacement synthesis deficient mutants such as Y64A M-MLV or F61WHIV or any other reverse transcriptase with impaired displacementsynthesis and increasing the Tm of the oligonucleotide to the RNA.

In any of these methods and compositions a thermostable DNA polymerasemay also be used, although with regard to RNA stability andhybridization kinetics of the primers this is not recommended in allsituations.

Especially for but not limited to optimal representation in a randomlyprimed cDNA library the random primers may be present in a concentrationfrom 50 nM to 100 μM, and more preferred at about 2.5 μM but can also beat least 300 nM. In preferred embodiments the ratio (w/w) of primer totemplate nucleic acids is between 5:1 and 1:1000, preferably between 2:1and 1:500, preferably between 1:1 and 1:300, preferably between 1:2 and1:250, preferably between 1:5 and 1:150, preferably between 1:10 and1:100, preferably between 1:12 and 1:50. The molar ratio of primer totemplate nucleic acids may be between 1,000:1 to 5,000,000:1, preferablybetween 5,000:1 to 1,000,000:1, between 10,000:1 to 500,000:1, orbetween 20,000:1 to 300,000:1. In one example, using 150 ng of mRNAstarting material and assuming an mRNA length of 500-5000 nt this wouldmean primers added at 2.5 μM final concentration are added in a molarexcess of 1:280,000-1:28,000. In preferred embodiments the molar or(w/w) ratio of primers to stoppers is between 2:1 and 1:10, preferablybetween 1:1 and 1:5, especially about 1:1.

However lowering the oligo concentration is possible e.g.: using 34 ngof mRNA starting material and assuming an mRNA length of 500-5000 ntthis would mean primers added at 300 nM final concentration are added ina molar ratio of 1:33-1:3.3, with the template being in a molar excess.A preferred reduced nucleotide concentration during the polymerizationreaction can help to reduce strand displacement and spurious secondstrand synthesis of the polymerase. In preferred embodiments the molaror (w/w) ratio of primers to stoppers is between 2:1 and 1:10,preferably between 1:1 and 1:5, especially about 1:1.

When mRNA is reverse transcribed an oligo dT primer can be added tobetter cover also the poly A tail of the mRNA. Optionally, the additionof the oligo dT primer can be omitted as it is part of the random primermix, although in a preferred embodiment to guarantee equalrepresentation of the mRNA it should be added. The length of oligo dTcan vary from 8 bases to 27 bases, but preferably an 25 nt long oligo dTprimer is used. Other types of primers with different composition can beused in place of oligo dT. Examples of such compositions include, butare not limited to, oligo dT where the 3′ base is A, or C, or G(anchored dT). Furthermore, other sequences or moieties that can basepair with poly A sequences of mRNA can also be used. An example, withoutlimitation, is deoxy uridine, (dU).

The oligo(dT) may consist essentially of between about 12 and about 25dT residues, and may be an anchored oligo(dT) molecule containing aterminal non-T nucleotide. The oligo(dT) may be oligo(dT) 18-25nt longor anchored equivalents thereof.

The oligo(dT) may be present in a concentration of between about 20 nMand about 1 μM, most preferably 500 nM are being used. The randomprimers may be between 5 and 15 nucleotides long, and may be randomhexamers with at least 3 LNAs or at least three 2′ Fluoro-modified basesat the 5′ site to efficiently stop strand displacement.

An additional feature is that depending on the concentration of therandom primer the average length of the product nucleic acids can beinfluenced. By increasing the concentration of the random primers theresulting elongated product size can be decreased. The desired fragmentsize depends on the subsequent application. If the desired amplifiednucleic acid portions should be below 300 nt (as is currently desiredfor next generation sequencing) the random primer concentrations shouldrange from 125 nM to 350 nM final concentration.

According to the methods of the invention the concentration of oligo dTcan be 44 nM to 750 nM, for example, or intermediate values. It isevident to those skilled in the art that various ratios of randomprimers and oligo dT can be used.

When mRNA is under investigation, preferably mRNA enriched RNA samplesshould be used. Several methods for mRNA enrichment are well documentedand known to those skilled in the art. The most commonly used is poly A+enrichment either by oligodT paramagnetic beads or oligodT cellulose[28]. A number of commercial kits for mRNA enrichment are available.Alternatively mRNA can be enriched by Terminator treatment (Epicentre).Additionally, several companies offer commercially available mRNAs froma number of organisms and tissues.

The concentration and combinations of modified-random primers and oligodT used in this invention provides efficient and representativeconversion of mRNA sequences into cDNA. This method provides superiorand non-biased conversion of mRNA sequences into cDNA regardless of thedistance from the 3′ end of the mRNA. Additionally it guarantees thatany RNA molecule is reverse transcribed only once by inhibiting thestrand displacement of the reverse transcriptase.

The present invention therefore relates to methods of increasing equalrepresentation of mRNA, and more particularly, to increasing theaccuracy of quantification of gene expression. Thus, the presentinvention provides improved cDNA synthesis useful in gene discovery,genomic research, diagnostics and identification of differentiallyexpressed genes and identification of genes of importance to disease.

In another embodiment oligonucleotide stoppers can be used tospecifically block the reverse transcription of unwanted such as but notlimited to high abundant or ribosomal transcripts during the reversetranscription process. This step can be used to suppress specifictemplates to generate a normalized mixture of products. A practical useis in suppressing abundant mRNA to generate a normalized cDNA library.Oligonucleotide stoppers that are used to suppress a template, i.e. arenot used to generate a well-defined elongation product are referredherein as blocking oligonucleotides or blockers. Blockingoligonucleotides are known from GB 2293238, US 2002/0076767 A1, U.S.Pat. No. 6,391,592 B1, WO 99/61661, WO 02/086155, U.S. Pat. No.5,849,497, WO 2009/019008 and can be employed according to the presentinvention. Preferably such blocking oligonucleotides would be highlyspecific for those transcripts which should be prevented from beingreverse transcribed, hence are preferentially longer than 15 nt. Longeroligonucleotides have a higher Tm, hence are harder to be displaced bythe reverse transcriptase. In a preferred embodiment the blockingoligonucleotides also have an increased Tm at the 5′ end by theintroduction of modifications such as but not limited to LNAs, ZNAs,PNAs or acridine. These blocking oligonucleotides will need to beblocked at the 3′ end to prevent them from being extended. Suchblockages include but are not limited to C3, C6, C12 spacers ordideoxynucleotides. The blocking oligonucleotides should preferentiallybe designed against a sequence that is located near the 3′ end of theRNA, but upstream of the poly A tail.

It is within the scope of the invention to combine the inventive methodsby using the above described blocking oligos together with the stranddisplacement synthesis deficient mutants such as Y64A M-MLV or F61W HIVor any other reverse transcriptase with impaired displacement synthesisas well as lowering the reaction temperature and using differentadditives to increase the binding of oligonucleotides to the RNA.

Alternatively, if the sequences of the high abundant transcripts are notknown, hence specific blocking oligos cannot be designed, the stranddisplacement stop can also be used for library normalization. A driverlibrary would be synthesized with SDS oligonucleotides (preferentiallysuch with increased local Tm at the 5′ end such as LNA, PNA or ZNAmodified oligonucleotides).

With a terminal transferase dideoxynucleotides can be added to thoseamplified nucleic acid portions in order to generate the blockingoligos. This library can then be hybridized to a tester template sample.High abundant transcripts will have an advantage and the single strandedamplified nucleic acid portion from the driver will be faster and moreefficient in hybridizing to the corresponding template. Productsynthesis can then be initiated using a primer, such as oligodT primerfor mRNA, and since most of the high abundant transcripts are hybridizedto blocking oligonucleotides, the low abundant transcripts have a fargreater chance of being reverse transcribed. If a template switch oroligo capping protocol is also used, the resulting cDNA of the lowabundant transcripts can even be inserted into a PCR reaction with theprimers corresponding to the oligo-dT primer and the template switch oroligo capping nucleotide. Alternatively selective terminal tagging (US2009/0227009 A1) can be used to tag the 3′ end of the newly synthesizedcDNA. Since high abundant transcripts are most likely to have neverreached full length due to the blocking oligonucleotides only lowabundant transcripts will be amplified in a PCR with primerscorresponding to the oligodT primer and the 3′ cDNA tag.

Alternatively, if the 3′ end of the cDNA is not tagged, an oligo-dTprimer with a double stranded T7 RNA polymerase promoter could be usedto prime the RT reaction of the normalized cDNA libraries and a linearamplification by in vitro transcription can be used.

Therefore a blocking oligonucleotide can be added to the elongation tostop unwanted sequence portions of a template molecule to be elongated.Pre-selected sequences of the unwanted template sequence are selectedfor use as blockers.

In preferred embodiments of the invention the template nucleic acid,e.g. the RNA template, is single stranded. In particular it is possiblethat the obtained template nucleic acid lacks a complementary strandover at least 30% of its length and/or lacks a complementary strand ofat least 100 nucleotides in length, preferably lacks a complementarynucleic acid over its entire length. The inventive method alsoencompasses separating possibly existing complementary strands from theinventive template nucleic acid strand and introducing the so purifiedtemplate nucleic acid without a complementary strand to the inventivemethod.

In particular preferred, the inventive one or more primers and one ormore stoppers bind to the template strand—and particularly not to acomplementary strand.

In a further aspect the present invention relates to the use of theabove described methods for generating one or more template nucleicacids to generate a sequence library comprising a mixture of amplifiednucleic acid portions of said template nucleic acids. Preferably, theamplified nucleic acid portions provide overlapping sequence portions ofthe template. This can e.g. be facilitated by using a multitude ofdifferent primers that generate various elongated products that in turnare used as amplified nucleic acid portions for the library.

The invention further provides a kit for generating amplified nucleicacid portions of a template nucleic acid as described herein or forgenerating a sequence library as described herein comprising a DNApolymerase, random oligonucleotide primers which comprise a modificationthat increases the Tm and random oligonucleotide stoppers that areunsuitable for nucleotide extension and comprise a modification thatincreases the Tm, optionally further one or more of reaction bufferscomprising Mn²⁺ or Mg²⁺, a ligase, preferably DNA ligase or RNA ligasewith DNA ligating activity, a crowding agent suitable for ligasereactions, like PEG. The ligase may also be a RNA ligase, especially aRNA ligase that has DNA ligating activity such as T4 RNA ligase 2.Crowding agents are inert molecules that can be used in highconcentrations and can be used to mimic the effects of macromolecularcrowding inside a cell. Examples are PEG (polyethylene glycol), PVP(polyvinylpyrrolidone), trehalose, ficoll and dextran. Crowding agentsare e.g. disclosed in U.S. Pat. No. 5,554,730 or U.S. Pat. No.8,017,339. The kit may alternatively comprise DNA polymerase and aligase and optionally any further compound mentioned above.

Further kits of the invention that are suitable for generating amplifiednucleic acid portions of a template nucleic acid comprise or contain a)random oligonucleotide primers which comprise a modification thatincreases the Tm and b) random oligonucleotide stoppers that areunsuitable for nucleotide extension and comprise a modification thatincreases the Tm. The kit may further comprise one or more of reactionbuffers comprising Mn²⁺ or Mg²⁺, a ligase, a crowding agent, such asPEG. The kit may also further comprise a DNA polymerase and/or a ligase.

Kits for use in accordance with the invention may also comprise acarrier means, such as a box or carton, having in close confinementtherein one or more container means, such as vials, tubes, bottles andthe like. The kit may comprise (in the same or separate containers) oneor more of the following: one or more reverse transcriptases, suitablebuffers, one or more nucleotides especially dNTPs, and/or one or moreprimers (e.g., oligo(dT, starters, stoppers, reverse complements, PCRprimers) for reverse transcription and subsequent PCR reactions. Thekits encompassed by this aspect of the present invention may furthercomprise additional reagents and compounds necessary for carrying outnucleic acid reverse transcription protocols according to the invention,such as oligodT beads or streptavidin coupled beads. Furthermore, theprimers or stoppers may be immobilized on a solid surface.

The present invention is further illustrated in the following figuresand examples, without being limited to these embodiments of theinvention.

DISCUSSION OF THE DRAWINGS

FIG. 1: Schematic representation of the principle problem the inventionseeks to solve.

a) Primers P1, P2 up to Pn are hybridized to a template RNA. Primer P2has hybridized to a more upstream (5′) position of the template RNA thanprimer P1 and more generally primer Pn has hybridized to a more upstreamposition on the template RNA than primer P(n-1). Or in other words,primer P1 has hybridized to a more downstream (3′) position on thetemplate than P2 and more generally P(n-1) has hybridized to a moredownstream position on the template RNA than Pn. Extension of eachprimer is initiated by a reverse transcriptase. b) When the reversetranscriptase while polymerizing the extension product of P2 reaches aprimer P3, primer P3 and its extension product get strand displaced bythe reverse transcriptase that continues to extend the primer P2extension product. The same is true for the extension product of P1 thatdisplaces primer P2 and its extension product. c) Therefore, when allextension products are finished, one cDNA copy of the template sequencebetween P1 and P2 is present, but two cDNA copies between P2 and P3 arepresent. This phenomenon leads to an overrepresentation of the 5′ endsof RNA that is primed multiple times, as it happens during standardreverse transcription using random primers such as random hexamers. Moregenerally speaking, when n primers have hybridized and were extended bythe polymerase to the 5′ end of the template RNA, then the 5′ end of theRNA will be represented n times while the 3′ end of the RNA will berepresented only once.

FIG. 2: Schematic representation of one embodiment of the invention tocreate a 5′-3′ balanced cDNA library and full length cDNA.

a) The hybridization of primers P1, P2 to Pn is reinforced by usinge.g.: locked nucleic acids (LNAs) to inhibit the strand displacementactivity of the reverse transcriptase. Here three modifications on thevery 5′ end of the primers are used. b) Now upon extending a primer thatlies more downstream on the template RNA the reverse transcriptasecannot displace the primer that has hybridized to a more upstreamposition on the template. Therefore, each portion of the RNA is onlyrepresented once as cDNA. In that manner no overrepresentation of RNA 5′ends will occur. c) When full length cDNA is desired the individualprimer extension products are ligated to yield d) one full length cDNAcopy of the template RNA.

FIG. 3: Schematic representation of creating a linker tagged short cDNAlibrary.

For many downstream analyses of cDNA libraries a universal linkersequence is needed either on one or both ends of the cDNA, to forinstance amplify the library or start a sequencing reaction. Here thepreparation of a library that has two linkers is shown. a) Primers P1,P2 to Pn have a 5′ universal linker sequence extension (L1). In additionstopper oligos S1, S2 to Sm are used that have a 3′ universal linkersequence extension (L2). The stopper oligos are also modified (e.g.LNAs) so they also cannot be displaced by the reverse transcriptase. b)In a second reaction step the 3′ end of the extension product is ligatedto the 5′ end of the stopper oligo. In that manner a cDNA library iscreated that has two linker sequences (L1, L2) available in order to c)amplify the whole library during e.g. a subsequent PCR.

FIG. 4: Schematic representation of creating a linker tagged short cDNAlibrary using an alternative stopper oligo concept.

As the error rate that is introduced into the cDNA library through amis-hybridization of the stopper oligo sequence is greater than if thisportion would have been transcribed by a polymerase, an alternativestopper oligo concept is shown. a) Here in comparison to FIG. 3 thestopper oligo Sm is extended on its 5′ side by an L2rc (reversecomplement) sequence. This L2rc is hybridized with L2 to form anadapter. b) Again as in FIG. 3 the reverse transcriptase is extendingthe primers Pn until they reach the stopper oligos Sm. During ligationthe extension product is now ligated to the L2 strand of the adapter. Inthis manner again a cDNA library is created that has two linkersequences (L1, L2) present on its ends. Here, however, no stopper oligosequence is introduced. Therefore, when sequencing from the L2 side ofthe library no ambiguity towards the identity of the first nucleotidesis introduced by a potential mis-hybridization of the stopper oligo. c)Finally, a PCR follows.

FIG. 5: Schematic representation of creating a linker tagged short cDNAlibrary using an alternative stopper oligo concept.

In an alternative concept starter and oligo form a heterodimer over acomplementary sequence in their respective linker sequences L1 and L2.Sequence gaps will be reduced since at any stop there is also apolymerization initiated by the starter. a) Starter/stopper hybrids areextended at the 3′ end of the starter sequence P1 to Pn until the nextstarter/stopper hybrid is encountered. Stopper oligos S1, S2 to Sm havea 3′ universal linker sequence extension (L2) which is in a hybrid withthe L1 sequence of the starter. It is sufficient if the stopper ismodified to prevent strand displacement although in an alternativesubset of such starter/stopper hybrids shown in FIG. 8e the modificationneeds to be located at the starter if the L2 sequence has no 5′ sequenceextension that is hybridizing to the template nucleic acid. b) In asecond reaction step the 3′ end of the extension product is ligated tothe 5′ end of the stopper oligo. In that manner a cDNA library iscreated that has two linker sequences (L1, L2) available in order to c)amplify the whole library during e.g. a subsequent PCR.

FIG. 6: Schematic representation of preferred primer modifications.

Depicted are primer modifications that are preferred. Thesemodifications can also be combined. a) General structure of primingoligo, with primer and linker sequence. When generating a randomlyprimed cDNA library the primer sequence is typically a random sequencesuch as, for instance, a random hexamer. b) The primer sequence partcontains modified nucleotides. The modification reduces or inhibits thestrand displacement activity of the reverse transcriptase. c) A reversecomplement (L1rc) is introduced that inhibits the L1 sequence part ofthe oligo to participate in the hybridization to the template RNAstrand. Therefore, a bias toward the L1 sequence is blocked. d) Toprevent the reverse transcriptase from associating with the linkeradaptors and hence lowering the efficiency of reverse transcription a 3′overhang is used at the 3′ end of L1rc. e) The L1rc sequence is modifiedto increase hybridization strength to the L1 sequence to further reducethe likelihood of bias in the library towards the L1 sequence. f)Blocking of all ends that do not participate in polymerase extension orligase reaction. g) The 5′ end of the L1 sequence and the 3′ end of theL1rc sequence are connected through a covalent bridge, e.g. a Cn spaceror a hairpin sequence.

FIG. 7: Schematic representation of preferred oligo stoppermodifications.

Depicted are stopper oligonucleotide modifications that are preferred.These modifications can also be combined. a) General structure ofstopper oligo, with stopper and linker sequence. The stopper sequence istypically a random sequence such as for instance a random nonamer. b)The stopper sequence part contains modified nucleotides. Themodification reduces or inhibits the strand displacement activity of thereverse transcriptase. c) A reverse complement (L2rc) is introduced thatinhibits the L2 sequence part of the oligo to participate in thehybridization to the template RNA strand. Therefore, a bias toward theL2 sequence is blocked. d) To prevent the reverse transcriptase fromassociating with the linker adaptors and hence lowering the efficiencyof reverse transcription, a 3′ overhang is used at the 3′ end of L2. e)The L2rc sequence is modified to increase hybridization strength to theL2 sequence to further reduce the likelihood of bias in the librarytowards the L2 sequence. f) Blocking of all ends that do not participatein polymerase extension or ligase reaction. g) The 5′ end of the L1sequence and the 3′ end of the L1rc sequence are connected through acovalent bridge, e.g. a Cn spacer or a hairpin sequence. h) The 5′ endof the stopper oligo is phosphorylated to be able to act as a donor inthe ligation reaction. Alternatively the 5′ end is adenlyated. i)Depicts the general structure of the stopper oligo used in thealternative oligo stopper concept in FIG. 4. The 5′ end of the L2sequence can be phosphorylated to be able to act as a donor in aligation reaction, or alternatively adenylated.

FIG. 8: Schematic representation of the most preferred oligo starter andstopper combinations.

Depicted are preferred starter/stopper combinations. Starters andstopper oligonucleotide contain a complementary (e.g. 14 nt) sequencestretch in their linker sequence, which allows them to hybridize witheach other therefore making the addition of additional reversecomplements obsolete. Starter and stopper designs from FIGS. 6 and 7 arestill valid. The starter is blocked by a 5′ OH. The stopperoligonucleotide is optionally blocked at the 3′ end by e.g.dideoxynucleotides, dioxynucleotides, spacers or inverted nucleotides.a) General structure of a stopper oligonucleotide in a hybrid with astarter oligonucleotide. Oligos are hybridized with each over e.g. a14nt stretch. Both starter and stopper oligonucleotides hybridize to thetemplate strand together. Starters and stoppers can have a hybridizationbase to the template nucleic acid of different lengths. The stopperoligonucleotide can have a longer or shorter hybrisation base comparedto the starter and vice versa. b) both the starter and stopper aremodified to inhibit strand displacement by the polymerase. c) here onlythe 5′ end nucleotides of the stopper are modified. This is enough asthe polymerase needs to be inhibited to strand displace only at the 5′end of the stopper. In effect a single modification (such as LNA or2′Fluoro) is enough. d) shows also a modification of the starter, as notall starters that are extended might be in a hybrid with a stopper thatalso has hybridized to the template. e) when the sequence of the stopperthat hybridizes to the template is reduced to 0 then the starter needsto also provide for the stop and therefore a modification that stopsstrand displacement is desirable. f) The L1 and L2 sequence can have apart that does not hybridize. g) shows an alternative configurationwhere the linker sequences have portions that hybridize to each otherand portions that don't. h) shows a configuration where the starter andstopper oligo are linked together forming in effect one oligonucleotide.Of course many more starter and stopper variations exist that can beused by someone skilled in the art.

FIG. 9: Schematic representation of the oligo starter and stopperstructures.

a) Starter oligonucleotides are depicted from 3′ to 5′ and b) stopperoligonucleotides are depicted from 5′ to 3′. The starter (a) consist of

(I) a priming sequence such as a random hexamer sequence binding to thetemplate strand on the 3′ side which preferably is modified to protectagainst strand displacement.(II) Optionally a barcode sequence can be located 5′ of the randompriming sequence. The barcode sequence preferentially consists of 3-9nucleotides, which allow a unique and specific identification of thelibrary. Such barcodes enable for instance to mix libraries fromdifferent samples and sequence them together on a flow cell. After thesequencing run the reads can be demultiplexed according to the specificbarcode.(III) The sequencing primer binding site. This is the sequence used tobind the sequencing primer during sequencing.(IV) a sequence for bridge amplification (e.g.: for Illumina NGSsequencing) or for attachment to a solid surface such as beads (e.g. forSOLiD NGS sequencing).(V) a sequence tag which provides a hybridization basis for starter andstopper oligo.A minimal starter can consist of a random priming part (I) and asequencing primer binding site (III),in which case a reverse complementas described in FIGS. 6c -g is preferred to prevent the sequencingprimer part from taking place in the hybridization process.Alternatively a starter can consist of a random priming part (I) and asequencing primer binding site (III), and a hybridization basis with thestopper, whereby III and V could be completely or partially identicale.g. if the sequencing primer binding site is complementary to asequence part III in the stopper region. Alternatively starters canconsist of I, II, III, and V, whereby again III and V could becompletely or partially identical e.g. if the sequencing primer bindingsite is complementary to a sequence part III in the stopper region.Starters can also consist of I, II, III, IV, and V.If a starter with a shorter linker sequence is used sequencescorresponding to sequence primer binding site (III), barcodes (II)and/or surface attachment (IV) can be introduced during amplification ofthe generated libraries e.g.: during a PCR reaction by introducing thesesequence tags with the PCR primers.Stopper oligonucleotides are depicted from 5′ to 3′in b.). The stopper(b) may consist of(I) a random sequence binding to the template on the 5′ side which ispreferably modified to protect against strand displacement.(II) Optionally a barcode sequence or a sequence that is reversecomplementary to the barcode sequence on the starter(II) can be located3′ of the random priming sequence. The barcode sequence preferentiallyconsists of 3-9 nucleotides, which allow a unique and specificidentification of the library.(III) The sequencing primer binding site.(IV) Optionally a sequence tag for surface attachment for bridgeamplification (e.g.: for Illumina NGS sequencing) or for attachment to asolid surface such as beads (e.g. for SOLiD NGS sequencing) as well as(V) a sequence tag which provides a hybridization basis for starter andstopper oligo. A minimal stopper can consist of a random priming part(I) and a sequencing primer binding site (III),in which case a reversecomplement as described in FIGS. 7c-g is preferred to prevent thesequencing primer part from taking place in the hybridization process.Alternatively a stopper can consist of a random priming part (I) and asequencing primer binding site(III), and a hybridization basis with thestarter, whereby III and V could be completely or partially identicale.g.: if the sequencing primer binding site is complementary to asequence part III in the stopper region. In an alternative embodimentthis minimal stopper may also lack the random sequence as shown in FIG.4 or 8 e. Alternatively stoppers can consist of I, II, III, and V,whereby again III and V could be completely or partially identical e.g.:if the sequencing primer binding site is complementary to a sequencepart III in the stopper region. Starters can also consist of I, II, III,IV, and V.

If a stopper with a shorter linker sequence is used sequencescorresponding to sequence primer binding site (III), barcodes (II)and/or surface attachment (IV) can be introduced during amplification ofthe generated libraries e.g.: during a PCR reaction by introducing thesesequence tags with the PCR primers.

FIG. 10: Stopping of strand displacement during reverse transcription.

a) Illustrates the assay set up for determining the ability of modifiedor non-modified oligonucleotides (Seq ID No: 5-8) to inhibit the stranddisplacement activity of the reverse transcriptases which are used inaddition to an oligo dT primer (Seq ID No: 9). The extension of thestrand displacement stop oligo is 35 nt (Seq ID No:10), the stranddisplacement stop product is 103 nt (Seq ID No:11) and the full lengthcDNA product is 138 nt (Seq ID No:12).

b) Shows the results of Example 1.

FIG. 11: Regulation of cDNA fragment size by amount of stop oligosinserted into the RT.

Shows the results of Example 2. cDNA fragments are obtained by randomprimed cDNA synthesis with or without strand displacement stop oligosand analyzed on an immunoblot.

FIG. 12: Stopping strand displacement during reverse transcription plusligation of the cDNA fragments to a full length product

Shows the results of Example 3. Here the strand displacement stop isinduced by an oligo with 3 LNAs at the 5′ end and the subsequentligation of the resulting 2 DNA fragments (35 nt, Seq ID No: 10 and 103nt, Seq ID No: 11) to the 138 nt full-length product (Seq ID No: 12)using either T4 DNA ligase (lane 3), T4 RNA ligase 2 truncated (lane 5)or a combination of both (lane 4). In lane 6 a control reaction omittingthe SDS oligo is shown. In lane 2 the SDS oligo was added, but no ligasewas added to the ligation reaction.

FIG. 13: Validation of SDS/Ligation on mRNA.

Shows the results of Example 4. The immunoblot shows a significantlength increase if the short strand displacement stop cDNA fragments areligated using T4 DNA ligase.

FIG. 14: Strand displacement stop during reverse transcription plusligation of the cDNA fragments to a full length product results in moreproduct of a selected cDNA.

The results of Example 5 are illustrated. qPCR results of a 5 kbfragment from Dynclhl (NM_030238) amplified from different reversetranscriptions (RTs) are shown. An RT using a SDS oligo (Seq ID No: 15)and an oligo dT primer (Seq ID No: 17) or oligo dT primer (Seq ID No:17) by itself was performed. T4 DNA ligase was added or in controlreactions not added and a qPCR was performed on the resulting cDNAs.Only in case of ligation the SDS oligos were able to produce the 5 kbfragment in a PCR reaction (primers Seq ID No:18 and Seq ID No:19).There is more PCR product in reactions performed on SDS/Ligation cDNA(i.e.: more cDNA template available) than in a regular oligo dT primedcDNA (compare lane 2 to lane 6 and 8, respectively).

FIG. 15: cDNA length comparison of SDS/ligation vs oligo dT priming on a15 kb cDNA.

FIG. 15 shows the results of Example 6, a qPCR assay designed to judgethe cDNA length generated in an RT reaction. Triangles: oligodT primedreverse transcription, squares: random hexamer primed transcription,circles: inventive transcription with stopped elongation at downstreamprimers plus ligation.

FIG. 16: Generation of a di-tagged DNA libray from mRNA.

Shows a DNA library generated using the SDS/ligation approach (see lane2), whereas the no ligation control stays empty (see lane 3). Withoutthe addition of RNA template some linker-linker byproducts can begenerated since the oligo dT primer will then serve as a template forhybridization (see lane 4). PCR no template controls are clean (see lane5).

FIG. 17: Discovery blot comparing a library preparation using the newstrand displacement stop and ligation protocol (SDS-ligation) with astandard mRNA Seq protocol (TruSeq™ RNA sample prep kit, Catalog #RS-930-20 01). Both were sequenced on an Illumina GAIIx sequencer(single read, 72bp). The X-axis shows the number of reads that uniquelymapped to the annotated genome vs the number of genes discovered on theY-axis. The comparison of the graphs shows that the SDS-ligationprotocol is feasible and actually needs less reads to detect the sameamount of genes than the standard protocol.

Abbreviations

-   qPCR: quantitative polymerase chain reaction-   SDS: strand displacement stop-   RT: reverse transcription or reverse transcriptase (depending on the    context)-   LNA: locked nucleic acid-   PNA: peptide nucleic acid-   rc: reverse complement-   Tm: melting temperature-   SNP: single nucleotide polymorphism-   CGH: comparative genomic hybridization-   CNV: copy-number variation-   PTO: phosphorothioate bond-   Phos: phosphorylation

Definitions

Starter: Starters are molecules that can prime a templated polymeraseextension reaction. Usually starters have an oligonucleotide sequencecommonly referred to as a primer. Starters can have 5′ sequenceextensions such as universal linker sequences described in FIGS. 6, 7, 8and 9.

EXAMPLES Example 1: Strand Displacement Stop of Reverse TranscriptasesUsing LNA Modified Oligonucleotides

Sequences: An asterix “*” denotes a phosphorothioate (PTO)bond, a plus “+” in front of a nucleotidedenotes a locked nucleic acid (LNA). “Phos” denotes a phosphorylation,SEQ ID No. 1: 5′-GCTAATACGACTCACTATAGTTGTCACCAGCATCCC-3′ SEQ ID No. 2:5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTCGAATGGGCCGCAGGA-3; SEQ ID No. 3:GCTAATACGACTCACTATAGTTGTCACCAGCATCCCTAGACCCGTACAGTGCCCACTCCCCTTCCCAGTTTCCGACTGTCCCCGGCCTCCTGCGGCCCATTCGAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ SEQ ID No. 4:5′-guugucaccagcaucccuagacccguacagugcccacuccccuucccaguuuccgacuguccccggccuccugcggcccauucgaaaaaaaaaaa aaaaaaaaaaaaaaaa-3′(RNA) SEQ ID No. 5: (Phos) (5′-+GGGCACTGTACG-3′) SEQ ID No. 6:(Phos) (5′-GGGCACTGTACG-3′) SEQ ID No. 7: (Phos) (5′-G*GGCACTGTAC*G-3′)SEQ ID No. 8: (Phos) (5′-+G+G+GCACTGTAC*G-3′) SEQ ID No. 9:5′-A*CGGAGCCTATCTATATGTTCTTGACATTTTTTTTTTTTTTTTTT TTTTTTTT*T*V-3′SEQ ID No. 10: 5′-GGGCACTGTACGGGTCTAGGGATGCTGGTGACAAC-3′ SEQ ID No. 11:5′-A*CGGAGCCTATCTATATGTTCTTGACATTTTTTTTTTTTTTTTTTTTTTTTTTTCGAATGGGCCGCAGGAGGCCGGGGACAGTCGGAAACTGGG AAGGGGAGT-3′SEQ ID No. 12: A*CGGAGCCTATCTATATGTTCTTGACATTTTTTTTTTTTTTTTTTTTTTTTTTTCGAATGGGCCGCAGGAGGCCGGGGACAGTCGGAAACTGGGAAGGGGAGTGGGCACTGTACGGGTCTAGGGATGCTGGTGACAAC-3′

To investigate the feasibility of inhibiting the strand displacementactivity of the reverse transcriptase under conditions that enableprimer annealing and cDNA polymerization, a proof of concept experimentis shown. For an outline of the assay setup see FIG. 10a , and forresults see FIG. 10 b.

Generation of in vitro transcribed 111 nt RNA template:

A 75bp fragment of GAPDH (NC_000072, 48 nt-122 nt) was PCR amplifiedwith primers containing either a T7 promoter sequence (Seq ID No: 1) ora T27 tail (Seq ID No: 2). The resulting PCR product (SEQ ID No: 3)served as template for T7 in vitro transcription using Epicentre'sAmpliScribe Flash T7 transcription kit. The in vitro transcribed 111 ntRNA (Seq ID No: 4) served as template for the strand displacement stopassay during reverse transcription.

cDNA Synthesis:

First-strand cDNA synthesis was carried out using MMLV-H from Promega(250 U/20 pl reaction). The strand displacement assay set-up is depictedin FIG. 10a . Primers for cDNA synthesis, LNA-modified oligos (1 LNA-G(Seq ID No: 5), unmodified oligos (Seq ID No: 6), PTO-modified oligos(Seq ID No: 7), 3 LNA-G oligos (Seq ID No: 8) or oligo dT (Seq ID No: 9)were ordered from either Micro-synth AG (Balgach Switzerland), orEurogentec (Seraing, Belgium). 800 ng of in vitro transcribed 111 nt RNA(Seq ID No: 4) and oligos (Seq ID No: 5-9; 50 nM oligodT primer SEQ IDNo:9 and 1.5 μM SEQ ID No: 5-8) were heated to 70° C. for 2 min with allrequired components (except for the reverse transcriptase) including:buffer (50 mM Bis-Tris-Methane pH 7.9, 75 mM KCl, 4 mM MgCl2, 0.6 MTrehalose and 7.5% glycerol), 0.5 mM each dNTP, 10 mM dithiothreitol(DTT), and slowly annealed by decreasing the temperature to 40° C. with30 sec holds at every 2° C. decrease. At 40° C. 250 units of reversetranscriptase were added and the temperature was slowly raised to 45° C.with 1 min holds at each 1° C. increase followed by a 30 minuteincubation at 46° C. Following first-strand synthesis, samples wereheated to 95° C. for 5 min in 0.1 M NaOH, neutralized with equalmolarities of HCl and purified by EtOH precipitation. After washing with75% EtOH the pellets were dissolved in 5 μl 10 mM Tris, pH 8.0 and mixedwith an equal volume of 100% formamide loading buffer, denatured at 95°C. for 2 min, cooled on ice and resolved by electrophoresis in a 15%acrylamide/7M urea.

Results are shown in FIG. 10 b.

In the different lanes the second downstream oligo (that has hybridizedto a more upstream portion of the template) was varied (Seq ID Nos: 5-8)using an oligo that had one LNA modification (Seq ID No: 5) in lane 2,no modification (Seq ID No: 6) in lane 3, one PTO (Seq ID No: 7) in lane4 or three LNA modifications (Seq ID No: 8) in lane 5 and no secondoligo in lane 6. Lane 1 and 7 show a size marker (10 bp marker,Invitrogen). It can be seen that the unmodified (lane 3) or the PTOmodified (lane 4) oligonucleotides are completely strand displaced byMMLV-H reverse transcriptase since only the full length product (Seq IDNo: 12) at 138nt is visible and no strand displacement stop product at103 nts (Seq ID No: 11). The second oligo has also been extended to 35nts ((Seq ID No: 10) and hence the 5′ end of the 111 nt RNA isrepresented twice (once in from of the full length product (138 nt) andonce by the 35 nt extension product of the second oligo). Introducing 1LNA at the 5′ sequence of the 2nd oligo already causes some stop ofstrand displacement (138 nt and 103 nt product are visible), while 3LNAs lead to an almost complete stop of strand displacement (no more 138nt product, just the 103 nt strand displacement stop product (Seq ID No:11).

Example 2: cDNA Fragment Size Regulation

This example shows that the size of a cDNA library that is generatedfrom mRNA can be regulated by the concentration of a random primer thatstops an elongation reaction. For results see FIG. 11.

RNA Isolation and Purification:

Total RNA from mouse liver was isolated using PeqLab Gold columns incombination with acidic phenol extraction (PeqLab, PEQLAB BiotechnologieGMBH, D-91052 Erlangen) according to manufacturer's recommendation. Theamount of RNA was measured by optical absorbance at 260 nm and checkedfor integrity on a formaldehyde agarose gel or an Agilent Bioanalyzer.

Terminator treatment of total RNA to enrich for mRNAs:

2-5 μg of total RNA was treated with Terminator™ 5′-Phosphate-DependentExonuclease (Epicentre Biotechnologies, Madison, Wis. 53713), accordingto manufacturer's instructions.

cDNA synthesis and immunoblotting:

cDNA synthesis was carried out in 20 μl reactions with 50 mM Tris-HCl(pH 8.3 at 25° C.), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 0.75 mM dNTPs withDigoxigenin-11-2′-deoxy-uridine-5′-triphosphate, alkali-stable. 135 ngof mRNA (terminator treated total RNA) was incubated at 70° C. for 2 minin the presence of primers and all the reaction components apart fromthe enzyme and the dNTPs. A slow annealing program was chosen with 30seconds holds at the following temperatures: 45° C., 43° C., 40° C., 38°C., 35° C., 30° C., 28° C. and 1 minute at 25° C. 200 U of MMLV-H, pointmutant (Promega) and dNTPs were added per 20 μl reaction and incubatedfor 2 min each at the following temperatures: 25° C., 28° C., 30° C.,and 35° C. be- fore a final extension at 37° C. for 10 min. Followingfirst-strand synthesis, samples were heated to 55° C. for 15 min in 0.1MNaOH, neutralized with equal molarities of HCl, purified by EtOHprecipitation. cDNA fragments were separated by formaldehyde agarose gelelectrophoresis (0.8%), transferred to Zeta-Probe GT Genomic BlottingMembranes (BioRAD) by electroblotting for 1 h at 50V according to “MiniTrans-BlotR Electrophoretic Transfer Cell” instruction manual (BioRAD),then crosslinked and by UV-light.

Membranes were equilibrated in 1× blocking buffer (100 mM maleicacid/150 mM NaCl, pH 7.5) for 5 min, before blocking unspecific bindingsites of the membrane in blocking solution (5% milk in blocking buffer)for 30 min. Anti Fab antibodies diluted 1:2,000 (Anti-Digoxigenin-AP FABfragments, Roche cat# 11 093 274 910); 30 ml blocking solution wereincubated for 30 min at room temperature under shaking. Membranes werewashed 2× for 15 min in 1× blocking buffer and then equilibrated for 5min in 1× staining buffer (0.1 M Tris-HCl, pH 9.5 (20° C.), 0.1 M NaCl).Staining in staining solution (BCIP®/NBT Liquid Substrate, 800 μl a 30ml 1× staining buffer) was done at room temperature (in the dark)overnight without shaking.

The results can be seen in FIG. 11.

In lane 1-5 an oligo (SDS-oligo) with three LNAs on its 5′ side (SEQ IDNo: 13: (Phos) (5′-+N+N+NNNN-3′)) was used in increasing concentrations(Lane 2: 0.25 μM; lane 3: 2.5 μM; lane 4: 25 μM, lane 5: 50 μM and lane6: 100 μM). In Lane 1 a control reaction with a non-modified randomhexamer (SEQ ID No: 14: (Phos) (5′-NNNNNN-3′)) at 2 μM is shown. Byincreasing the amount of SDS oligos the size of the generated cDNAfragments can be decreased.

Example 3: Ligation Using an Artificial Template

The example shows that an extension product of a more upstream primer(P1) can be stopped by a more downstream primer (P2) that has three LNAmodifications on its 5′ end and that the extension product of theupstream primer (P1) can by ligated to the downstream primer (P2) whenall are in a hybrid with the template. For an overview of the sequencesinvolved see FIG. 10a . For results see FIG. 12. Reverse transcriptionwas carried out using oligos, template and conditions as described inExample 1.

After the RT the samples were ethanol precipitated and inserted into a15 pl ligation reaction with 1 mM HCC, 20% PEG-8000, 30 mM Tris-HCl (pH7.8 at 25° C.), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP and 1.5 μl of T4 DNAligase (1-3 Weiss units/pl, Promega) and/or 1 μl of T4 RNA ligase 2truncated (10 units/μl, NEB) and incubated 2 h at 37° C. Unligated smallfragments and remaining oligos were removed by PEG precipitation.Therefore the volume of the reaction was increased to obtain 11.5% finalPEG concentration and 2 μl of linear polyacrylamide (10 mg/ml) wereadded as carrier. Reactions were thoroughly vortexed beforecentrifugation at 20,000×g for 15 min at 18° C. The pellet was washed 2×in 75% EtOH, dissolved in 5 μl 10 mM Tris (pH 8.0), mixed with an equalvolume of 100% formamide loading buffer, denatured at 95° C. for 2 min,cooled on ice and loaded onto a 15% acrylamide/7M urea gel. The resultscan be seen in FIG. 12. In lane 6 the artificial RNA was reversetranscribed using only an oligo dT primer (SEQ ID No: 9). When an SDSoligo (SEQ ID No: 8) is added in addition to the oligo dT primer (SEQ IDNo: 9), there is a complete stop of strand displacement as seen by theappearance of a 103 nt SDS product (Seq ID No: 11, see FIG. 12, lane 2).Furthermore a 35 nt SDS oligo extension product (Seq ID No: 10) isgenerated. T4 DNA ligase (FIG. 12, lane 3) or T4 RNA ligase 2, truncated(FIG. 12, lane 5) as well as a combination of both enzymes (FIG. 12,lane 4) were tested for their efficiency to ligate the two cDNAfragments in an RNA hybrid. T4 RNA ligase 2, truncated, is deficient ofan adenlyation function and hence can only ligate already adenylatedoligos i.e.: oligos that were previously adenylated by T4 DNA ligase inthe hybrid. T4 RNA ligase 1 was not used due to the preferred ligationof single stranded molecules which would then also result in ligation ofnon-hydridized oligos preferentially to the RNA template. As can be seenin lane 3-5 the stopped products (103 nt, Seq ID No: 11) and the SDSoligo extension product (35 nt, Seq ID No: 10) can be ligated in the RNAhybrid, resulting in a full-length 138nt cDNA (Seq ID No: 12).

Example 4: Ligation of cDNA Fragments Synthesized from Poly A-mRNA

The example shows that by ligating short cDNA fragments that weregenerated by the inventive method a size shift occurs on the immunoblot,indicating that longer cDNA was created by the ligation process. Formethods see Example 2 and 3. For results see FIG. 13. Poly A selectedmRNA (mouse liver polyA+mRNA, Stratagene) was used as a template. ShortcDNA fragments (see FIG. 13, lane 1, fragments between 100-700 nt) thatwere generated using combination of oligo dT primer (SEQ ID No: 9) and arandom dodecamer with 3 LNA modified nucleotides (SEQ ID No: 15: (Phos)(5′-+G+G+GHHHNNNNNN-3′)) were ligated in the RNA hybrid using T4 DNAligase, which results in long (full-length) cDNAs even longer than 6,000nts (see FIG. 13, lane 2).

Example 5: In a Gene-Specific qPCR the SDS/Ligation ReverseTranscription Yields More Product than a Regular oligodT Primed RT

A gene-specific PCR was carried in a 20 μl reaction containing 1 μl cDNA(synthesized from 800 ng total RNA, dissolved in 42 μl 10 mM Tris, pH8.0 after purification), 50 mM Tris-Cl pH 9.2, 16 mM ammonium sulfate,0.1% Tween 20, and 5.1 mM MgCl₂, 1.5M Betaine, 1.3% DMSO, 0.5×SYBRGreenI, 0.2 mM of each dNTP, 0.3 μM of each primer (SEQ ID No: 18:5′-CTGGATGAATGGCTTGAGTGT-3′ and SEQ ID No: 19:5′-GCAACTCCACGCTCATAGAAG-3′, primers designed for NM_030238), 0.8 unitsKlenTaq AC polymerase and 0.2 units Pfu polymerase. Samples weredenatured at 95.8° C. for 15 sec, and cycled 20 times at 95.8° C. for 15sec, 55° C. for 30 sec, 74° C. for 20 min (ramp speed at ABI9700: 50%).Subsequently, 19 cycles at 95.8° C. for 15 sec, 58° C. for 30 sec, 74°C. for 20 min (ramp speed at ABI9700: 10%) with a final extension stepat 72° C. for 3 min followed. PCR products were purified using silicacolumn and were loaded onto a 0.7% agarose gel. Results are shown inFIG. 14. Lane 8 shows the 5096bp PCR product generated from a cDNAsynthesized with an oligo dT primer (Seq ID NO: 17:5′-G*GCGTTTTTTTTTTTTTTTTTT*V-3′). As a control oligodT primed cDNA wasalso subjected to the ligation protocol that is usually applied for thestrand displacement stop oligos (see lane 6). When Seq ID No: 15 (SDSoligos) and Seq ID No: 17 (oligo dT) were used to prime the RT reactionfollowed by a ligation protocol as described in Example 3, the amount ofPCR product generated from such a cDNA was even more than from a regularoligo dT primed cDNA (compare FIG. 14, lane 2 to lane 6 and 8,respectively). This can be explained by the SDS oligos preventingsecondary structure formation due to hybridization, whereas thosesecondary structures lead to premature polymerization stop events ifonly an oligo dT primer is used. With the SDS/ligation protocol the RTis started at multiple places and the resulting cDNA fragments are thenligated to give full-length cDNA products or in this case the selected 5kb fragment from a randomly chosen specific transcript. Without ligationno PCR product is generated in the subsequent PCR of cDNA synthesizedwith SDS oligos (see lane 4). This clearly shows that the stranddisplacement was actually stopped and the generated cDNA fragments werenot connected, hence no PCR product can be obtained, since there is notemplate containing both PCR primer binding sites. Lane 3, 5, and 7 arecontrols were no reverse transcriptase was added to the RT reaction,hence showing there was no genomic DNA contamination and that the SDSoligos do not result in any unspecific background.

Example 6: Long Transcripts are More Efficiently Reverse TranscribedUsing a SDS/Ligation RT Protocol

A long transcript was chosen (ubiquitin protein ligase E3 componentn-recognin 4; Ubr4; NM_001160319) and 200 bp amplicons were designedalong the cDNA. Amplicons were spaced approximately 2 kb apart from eachother (primers Seq ID No: 20-29).

SEQ ID NO. 20: 5′-CCTTCCAGGAGGAGTTCATGCCAGT-3′ SEQ ID NO. 21:5′-CACACGGAGAGATGAATGAGGGGAGA-3′ SEQ ID NO. 22:5′-GCCTTCATGGCTGTGTGCATTGA-3′ SEQ ID NO. 23:5′-CATCCTGCCCTGTAGAAAGTCCTCTTG-3′ SEQ ID NO. 24:5′-CCAGTGTCACAAGTGCAGGTCCATC-3′ SEQ ID NO. 25:5′-GCGGTCAGCTTTGTCCAGAAGTGTGT-3′ SEQ ID NO. 26:5′-GTAAGATGGTGGATGGGGTGGGTGT-3′ SEQ ID NO. 27:5′-TCGCTCTGAAATGCTGACTCCTTCA-3′ SEQ ID NO. 28:5′-ACCCAGGTTCTACTGCGTCCTGTCC-3′ SEQ ID NO. 29:5′-CCTCCAGGGCTGTCACGTTCTTCTT-3′

The delta Ct between the more 3′ amplicons and the most 5′ amplicon(complementary to the 3′ end of the mRNA, close to the poly A tail) isused to calculate the fold difference (according to Pfaffl [34]) andhence shows the relative decrease in cDNA generated over the length ofthe mRNA template. Oligo dT (Seq ID No: 17) primed cDNA was compared toa cDNA primed with Seq ID: 15 and Seq ID No: 14 followed by a ligationof the resulting cDNA fragments in the RNA hybrid. Each of the reactionwas performed in triplicates and the means are depicted in FIG. 15.Circles depict values that were measured using SDS-oligos (dashed line);Squares when random hexamers were used (continuous line) and triangleswhen oligo dT was used (dotted-dashed line). Only the SDS/ligationprotocol guarantees a more equal cDNA synthesis over the length of thetranscript having an almost perfect 3′ to 5′ ratio of 1.

Oligo dT primed cDNA (triangles, dotted-dashed line) shows a steadydecline in cDNA generated over the length of the 15 kb mRNA. Onlyapproximately 10% of the originally started cDNA synthesis reaches 6 kb.Random priming without strand displacement leads to anoverrepresentation of the 5′ end of the mRNA (squares, continuous line).

Example 7: Generation of a Di-Tagged DNA Library from mRNA

0.11 μM Biotin tagged oligo dT primer (SEQ ID NO. 30: (Biotin-TEG)(5′-TTTTTTTTTTTTTTTTTTTTTTTTTTT-3′), 2.5 μM tagged primer (SEQ ID NO.31: (C12-Spacer) (5′-TCCCTACACGACGCTCTTCCGATCTGACTG+G+G+GNNN-3′)+2.5 μMreverse complement to the primer tag (SEQ ID NO. 32: (C3-Spacer)(5′-CAGTCAGATCG+GAA+GA+GC+GTC+GT+GTAGGGA-3′) (C3-Spacer)), 5 μM taggedstopper (SEQ ID NO. 33: (Phos)(5′-+G+G+GHHNNNNAGATCGGAAGAGCGGTTCAGCAGGA-3′) (C3Spacer))+5 μM reversecomplement to the stopper tag (SEQ ID NO. 34: (C12-spacer)(5′-TCCT+GCT+GAACC+GCTCTTCC+GATC-deoxyT-3′),deoxyT denotes a 3′ blockeddeoxyT), were hybridized to 150 ng of polyA+mRNA (BioCat Heidelberg,Germany) hybridized in Tris, pH 7.0 (70° C., 1 min, then slowly cooledon ice). Assuming an mRNA length of 500-5,000 nt this would mean thatthe starters are added in a molar excess of 1:280,000-1:28,000, whereasthe stoppers are added in a molar excess of 1:560,000-1:56,000. Thehybridized nucleic acids were then bound (20 min at room temperature) topre-washed 1.1 μl Streptavidin coated Dynabeads (M-280, 10 mg/ml).Non-hybridized nucleic acids were washed away (4 washes according to themanufacturer's instructions). Afterwards RT buffer was added to a finalconcentration of 1× (50 mM Tris-HCl (pH 8.3 at 25° C.), 75 mM KCl, 3 mMMgCl₂, 10 mM DTT), 0.5 mM dNTPs, 200 Units MMLV-H as well as 8U of T4DNA ligase plus 10% PEG and 0.4 μM ATP. The reversetranscription-ligation reaction was performed on beads by heating thereaction slowly raising the temperature (25° C. for 2 min, 28° C. for 1min, 31° C. for 1 min, 34° C. for 1 min) before incubating for 2 hoursat 37° C. Again the beads were washed (4×), before hydrolyzing the RNA(55° C. for 15 min in 0.1 N NaOH). After neutralization with 0.1 N HCl,samples were precipitated, dissolved in 20 μl and 4 μl were theninserted into an Illumina qPCR (primers: SEQ ID NO. 35:5′-A*ATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ andSEQ ID NO. 36:5′-C*AAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATC*T-3′).Results are shown in FIG. 16. A library smear is generated using theSDS/ligation approach (see lane 2), whereas the no ligation controlstays empty (see lane 3). Without the addition of RNA template somelinker-linker byproducts can be generated since the oligo dT primer willthen serve as a template for hybridization (see lane 4). PCR no templatecontrols are clean (see lane 5).

Example 8: SDS-Ligation Samples were Prepared as Described in Example 7

For comparison a standard mRNA-Seq library was prepared according to anIllumina library prep protocol (TruSeq™ RNA sample prep kit, Catalog #RS-930-20 01). Both libraries were sent for NGS sequencing on anIllumina GAIIx machine (single read, 72 bp). To compare the performanceof both libraries discovery blots were calculated showing the number ofdetected genes in relation to a given number of reads that mappeduniquely to the annotated genome. The discovery blots are shown in FIG.17. They show the feasibility of the SDS-ligation protocol that actuallyoutperforms the standard m-RNA Seq library preparation protocol.

Example 9

Di-tagged libraries were prepared from universal human reference RNA(Agilent Technologies, Catalog # 740000) spiked in with ERCC RNA spikein control mix (Catalog # 4456740) according to the manufacturer'sinstruction. Two NGS sample preparation methods were used: eitherScriptSeq V2 kit (cat# SSV21106, Epicentre, Wis.) according to themanufacturer's instructions or a sample preparation as described inexample 7 with the following modifications: oligodT25 magnetic beadsfrom Dynazyme (taken from the mRNA direct kit Catalog # 610-12) wereused as well as LNA-N modified

starters SEQ ID No. 37: (5Sp9) (5′-TCCCTACACGACGCTCTTCCGATCTAGC+N+N+NNNN-3′) and stoppers SEQ ID No. 38:(phos) (5′-+N+N+NNNNNNNAGATCGGAAGAGCGGTTCAGCAGG A-3′) (C3 spacer).

In Table 1 the results of the NGS sequencing run are listed.

TABLE 1 Determination of library strandednes on ERCC spike intranscripts.  

 

indicates data missing or illegible when filed

The strandedness (conservation of strand information) was determined forboth methods using the ERCC RNA spike in controls. These controlsprovide an absolute measure of the strandedness since they only exist inone orientation i.e.: there is no antisense. Should a method detectantisense transcripts of the ERCCs it is a direct measure of theinherent error rate of said method. The strandedness is calculated asthe median of 1000 reads/ERCC for both methods. The SDS-ligation methodshowed 100% strand specificity with no reads going into the wrongdirection whereas with ScriptSeq the strandedness is determined to be97.52%.

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1-37. (canceled)
 38. A method of ligating DNA molecules that are in ahybrid with an RNA molecule, the method comprising providing DNAmolecules that are in a RNA:DNA hybrid with an RNA molecule, andligating said DNA molecules to each other with a double strand specificligase.
 39. The method of claim 38, further comprising changing aconformation of the RNA:DNA hybrid to a double helix confirmation of aDNA:DNA helix.
 40. The method of claim 38, wherein the step of ligatingsaid DNA molecules is conducted in the presence of PEG, Tween-20, orNP-40.
 41. The method of claim 40, wherein said PEG, Tween-20 or NP-40is in an amount sufficient to change the RNA:DNA hybrid to a DNA:DNAhelix confirmation.
 42. The method of claim 40, wherein the step ofligating said DNA molecules is conducted in the presence of PEG and PEGis used at a concentration between 12% and 25% (v/v).
 43. The method ofclaim 38, wherein the step of ligating said DNA molecules is conductedwith a ligase selected from the group consisting of T4 RNA ligase, T4DNA ligase, T4 RNA ligase 2, Taq DNA ligase, and an E. coli ligase. 44.The method of claim 38, wherein the step of ligating said DNA moleculesis conducted in the presence of pyrophosphase.
 45. The method of claim38, wherein said DNA comprises LNA.
 46. The method of claim 38, whereinsaid DNA molecules comprise a linker that is not hybridized to the RNAmolecule.
 47. A method for generating an amplified nucleic acid portionof a template nucleic acid, which comprises: obtaining template nucleicacid; annealing at least one oligonucleotide primer to said templatenucleic acid; annealing at least one oligonucleotide stopper and/orfurther primer to said template nucleic acid; and elongating the atleast one oligonucleotide primer in a template specific manner until theelongating product nucleic acid reaches the position of an annealedoligonucleotide stopper or further primer, whereby the elongationreaction is stopped, wherein in said elongation reaction said optionaloligonucleotide stopper is not elongated and/or said furtheroligonucleotide primer is elongated in a template specific manner;wherein the elongated product nucleic acid is ligated to the 5′ end ofsaid oligonucleotide stopper or further primer according to the methodof claim 1.